• 센서학회지
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• 제30권4호
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• pp.229-235
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• 2021

This study presents a simple approach for the assembly of a free-standing conductive electronic nanofilm of single-walled carbon nanotubes (SWNTs) suitable for enzymatic electrochemical biosensors. A large-scale SWNT electronic film was successfully produced by the dialysis of p-Terphenyl-4,4''-dithiol (TPDT)-treated SWNTs. Furthermore, Horseradish peroxidase (HRP) was immobilized on the TPDT-SWNT electronic film, and the enzymatic detection of hydrogen peroxide (H₂O₂) was demonstrated without mediators. The detection of H₂O₂ in the negative potential range (-0.4 V vs. Ag/AgCl) was achieved by direct electron transfer of heme-based enzymes that were immobilized on the TPDT-SWNT electronic film. The SWNT-based biosensor exhibited a wide detection range of H₂O₂ from 10 µM to 10 mM. The HRP-doped SWNT electronic film achieved a high sensitivity of 342 ㎛A/mM·cm² and excellent selectivity against a variety of redox-active interfering substances, such as ascorbic acid, uric acid, and acetaminophen.

• Journal of Microbiology and Biotechnology
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• 제31권3호
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• pp.464-474
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• 2021

Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37℃. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H2O2) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.

• Mycobiology
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• 제50권2호
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• pp.121-131
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• 2022

The rare edible and medicinal fungus Antrodia cinnamomea has a substantial potential for development. In this study, Illumina HiSeq 2000 was used to sequence its transcriptome. The results were assembled de novo, and 66,589 unigenes with an N50 of 4413 bp were obtained. Compared with public databases, 6,061, 3,257, and 2,807 unigenes were annotated to the Non-Redundant, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes databases, respectively. The genes related to terpene biosynthesis in the mycelia of A. cinnamomea were analyzed, and acetyl CoA synthase (ACS2 and ACS4), hydroxymethylglutaryl CoA reductase (HMGR), farnesyl transferase (FTase), and squalene synthase (SQS) were found to be upregulated in XZJ (twig of C. camphora) and NZJ (twig of C. kanehirae). Moreover, ACS5 and 2,3-oxidized squalene cyclase (OCS) were highly expressed in NZJ, while heme IX farnesyl transferase (IX-FIT) and ACS3 were significantly expressed in XZJ. The differential expression of ACS1, ACS2, HMGR, IX-FIT, SQS, and OCS was confirmed by real-time quantitative reverse transcription PCR. This study provides a new concept for the additional exploration of the molecular regulatory mechanism of terpenoid biosynthesis and data for the biotechnology of terpenoid production.

• 대한본초학회지
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• 제34권2호
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• pp.1-14
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• 2019

Objective : This study was designed to evaluate the protective effect of Rehmanniae Radix Crudus (RC) in 150 mM HCl/ethanol induced acute gastritis mice. Methods : ICR mice were divided into 5 groups (normal group, control group, 10 mg/kg sucralfate treated group, 50 mg/kg RC treated group, 100 mg/kg RC treated group, n=8). Normal group was not take any treatment. Control group induced gastritis 1 hour after ingestion of distilled water. 10 mg/kg sucralfate induced group was induced gastritis 1 hour after ingestion of distilled of sucralfate 10 mg/kg. 50 mg/kg and 100 mg/kg RC treated groups were induced gastritis 1 hour after ingestion of distilled of RC 50 mg/kg and 100 mg/kg. After 1 hour of gastritis induction, removed the stomach tissue. We examined histological observations, oxidative stress biomarkers, antioxidant proteins, inflammatory mediators and cytokines. Results : In this study, the RC treatment group showed gastritis and gastric ulcer inhibition, and the area of injury decreased. The oxidative stress biomarkers such as reactive oxygen species (ROS) and peroxy nitrite ($ONOO^-$) in the serum were reduced in the RC treated group. Inaddition, antioxidant proteins (nuclear factor erythroid 2-related factor 2, Heme oxygenase 1) were increased in RC treated group, and the expression of inflammatory mediators and cytokines induced by nuclear factor-kappa B activation was inhibited. Conclusion : According to the results, RC may have an excellent inhibitory effect on acute gastritis and gastric ulcer.

• 대한본초학회지
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• 제37권5호
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• pp.83-88
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• 2022

Objectives : Jakyakgamcho-tang (JGT) has been traditionally used to treat muscular convulsion and pain in South Korea. According to recent studies, JGT has been reported to have anti-depression, anti-inflammation, anti-oxidative, anti-diabetics, anti-spasm and analgesic effects, but studies on its anti-neuroinflammatory and neuroprotective effect have not been deeply conducted. Thus, we investigated the anti-neuroinflammatory activity of JGT on lipopolysaccharide (LPS)-stimulated mouse microglia cells. Methods : To investigate the anti-neuroinflammatory effects of JGT on BV2 microglial cells, we examined the production of nitric oxide (NO) using griess assay, and mRNA expressions of pro-inflammatory cytokines such as interleukin (IL)-1𝛽, IL-6, and tumor necrosis factor (TNF)-𝛼 using real time RT-PCR. Furthermore, to determine the regulating mechanisms of JGT, we investigated the heme oxygenase (HO)-1 by real time RT-PCR. Results : Pre-treatment of JGT effectively decreased NO production in LPS-stimulated BV2 cells at concentrations without cytotoxicity. Additionally, JGT significantly suppressed the production of IL-1𝛽, IL-6, and TNF-𝛼 in LPS-stimulated BV2 cells. Furthermore, JGT activated the HO-1 expression, which is one of the immunomodulatory signaling molecules. And the abolishment of HO-1 by tin protoporphyrin IX (SnPP, the HO-1 inhibitor) reversed the anti- inflammatory activity of JGT in LPS-stimulated BV2 cells. Conclusions : Our results suggest that the JGT has anti-neuroinflammatory effect through the activation of HO-1 in LPS-stimulated BV2 cells. Thereby, JGT could expected to be used for the prevention and treatment of neurodegenerative disease related to neuroinflammation.

• Molecules and Cells
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• 제45권8호
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• pp.575-587
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• 2022

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metal-free porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible "bulge" loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

• Biomolecules & Therapeutics
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• 제30권5호
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• pp.447-454
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• 2022

Few studies have evaluated the role of autophagy in the development of oxaliplatin (OXT) resistance in colon cancer cells. In this study, we compared the role of autophagy between SNU-C5 colon cancer cells and OXT-resistant SNU-C5 (SNU-C5/OXTR) cells. At the same concentration of OXT, the cytotoxicity of OXT or apoptosis was significantly reduced in SNU-C5/OXTR cells compared with that in SNU-C5 cells. Compared with SNU-C5 cells, SNU-C5/OXTR cells exhibited low levels of autophagy. The expression level of important autophagy proteins, such as autophagy-related protein 5 (Atg5), beclin-1, Atg7, microtubule-associated proteins 1A/1B light chain 3B I (LC3-I), and LC3-II, was significantly lower in SNU-C5/OXTR cells than that in SNU-C5 cells. The expression level of the autophagy-essential protein p62 was also lower in SNU-C5/OXTR cells than in SNU-C5 cells. In SNU-C5/OXTR cells, the production of intracellular reactive oxygen species (ROS) was significantly higher than that in SNU-C5 cells, and treatment with the ROS scavenger N-acetylcysteine restored the reduced autophagy levels. Furthermore, the expression of antioxidant-related nuclear factor erythroid 2-related factor 2 transcription factor, heme oxygenase-1, and Cu/Zn superoxide dismutase were also significantly increased in SNU-C5/OXTR cells. These findings suggest that autophagy is significantly reduced in SNU-C5/OXTR cells compared with SNU-C5 cells, which may be related to the production of ROS in OXT-resistant cells.

• Journal of Ginseng Research
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• 제47권2호
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• pp.311-318
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• 2023

Background: The beneficial effects of compound K (CK) on different chronic diseases have been shown to be at least related to antioxidant action. Nevertheless, since its antioxidant activity in human retinal pigment epithelial (RPE) cells is still unknown, here we investigated whether CK alleviates oxidative stress-stimulated damage in RPE ARPE-19 cells. Methods: The cytoprotective consequence of CK in hydrogen peroxide (H₂O₂)-treated cells was evaluated by cell viability, DNA damage, and apoptosis assays. Fluorescence analysis and immunoblotting were performed to investigate the inhibitory action of CK on reactive oxygen species (ROS) production and mitochondrial dysfunction. Results: H₂O₂-promoted cytotoxicity, oxidative stress, DNA damage, mitochondrial impairment, and apoptosis were significantly attenuated by CK in ARPE-19 cells. Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation level and its shuttling to the nucleus were increased, which was correlated with upregulated activation of heme oxygenase-1 (HO-1). However, zinc protoporphyrin, a blocker of HO-1, significantly abrogated the preventive action of CK in H₂O₂-treated ARPE-19 cells. Conclusion: This study indicates that activation of Nrf2/HO-1 signaling by CK plays an important role in rescuing ARPE-19 cells from oxidative cellular damage.

• 대한통합의학회지
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• 제11권3호
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• pp.159-169
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• 2023

Purpose : Though other Citrus spp. have reported their anti-inflammatory and antioxidative activities in previous studies, the biological activity of Kiyomi (Citrus unshiu × C. sinensis) has not been reported yet. Therefore, this study attempted to analyze the anti-inflammatory mechanisms of Kiyomi leaf ethanol extract (KLEE) in lipopolysaccharide (LPS) stimulated RAW 264.7 cells. Methods : The cytotoxic effect of KLEE in RAW 264.7 cells was determined by WST-1 assay. Bacterial endotoxin, the concentration of nitric oxide (NO) was analyzed by the Griess reaction. In addition, Western blot analysis was applied to measure the protein expression level of inducible NO synthase (iNOS). The phosphorylated status of the critical inflammatory transcription factor, nuclear factor (NF)-𝜅B, and its upstream signaling molecules, phosphoinositide 3-kinase (PI3K)/Akt as well as mitogen-activated protein kinases (MAPKs), were also measured by Western blot analysis. Results : KLEE was not cytotoxic up to a concentration of 200 ㎍/㎖, and protein expression levels of iNOS and cyclooxygenase (COX)-2, enzymes that counteract NO and prostaglandin (PG) E2 production, were inhibited by KLEE treatment. The phosphorylated status of PI3K/Akt as well as MAPKs including extracellular regulated kinase (ERK), c-jun NH2kinase (JNK), and p38, were significantly attenuated by KLEE treatment in LPS stimulated RAW 264.7 cells. Moreover, one of phase II enzymes, heme oxygenase (HO)-1 which has known for its anti-inflammatory capacity, was strongly induced by KLEE treatment. Conclusion : Consequently, KLEE treatment significantly attenuated the production of NO as well as the expression levels of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. The inflammatory transcription factor, NF-𝜅B, as well as its upstream signaling molecules, PI3K/Akt and MAPKs, were also diminished by KLEE treatment with statistical significance in LPS-stimulated RAW 264.7 cells. These results suggest that KLEE might be a promising candidate for the attenuation of inflammatory disorders.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.387-397
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• 2023

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.