• Fisheries and Aquatic Sciences
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• v.14 no.4
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• pp.303-310
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• 2011

We characterized a cytoplasmic actin gene locus in greenling Hexagrammos otakii (Scorpaeniformes). Genomic clones isolated from the greenling DNA library contained two homologous cytoplasmic actin gene copies (HObact2.1 and HObact2.2) in a tail-to-head orientation. Their gene structure is characterized by six translated exons and one non-translated exon. Exon-intron organization and the nucleotide sequences of the two actin gene isoforms are very similar. However, only the HObact2.1 isoform contains microsatellite-like, dinucleotide repeats in the 5'-flanking region (named HOms2002) and intron 1 following the non-translated exon 1 (named HOms769). One microsatellite locus (HOms769) was highly polymorphic while the other (HOms2002) was not. Based on bioinformatic analysis, different transcription factor binding motifs are related to stress and immune responses in the two actin isoforms. Semiquantitative and real-time reverse transcription-PCR assays showed that both isoform transcripts were detectable ubiquitously in all the tissues examined. However, the basal expression levels of each isoform varied across tissues. Overall, the two isoforms showed a similar, but not identical, expression pattern. Our data suggest that the cytoplasmic actin genes may be the result of a recent duplication event in the greenling genome, which has not experienced significant subfunctionalization in their housekeeping roles.

• Journal of Photoscience
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• v.9 no.2
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• pp.98-101
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• 2002

Euglena gracilis abruptly changes its swimming direction after a sudden increase or decrease in incident light intensity, that is, step-up or step-down photophobic responses, resulting in photoavoidance or photoaccumulation, respectively. To identify the photoreceptor molecules for these UV-A/blue-light type photobehaviors, we purified a flavoprotein from isolated putative photosencory organelles (PFBs) of Euglena. The purified flavoprotein, which noncovalently bound flavin adenine dinucleotide (FAD), seemed to be a heterotetramer of alpha- and beta-subunits. Predicted amino acid sequences of each of the subunits were similar to each other and contained two FAD-binding domains each followed by an adenylyl cyclase catalytic domain. The purified flavoprotein actually showed adenylyl cyclase activity, being drastically elevated by blue-light irradiation. Suppression of gene expression of the flavoprotein (Photoactivated Adenylyl Cyclase, PAC) by RNA interference (RNAi) caused loss of the step-up photophobic response, demonstrating that PAC actually mediates photoavoidance of Euglena.

• Journal of Ginseng Research
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• v.44 no.1
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• pp.161-167
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• 2020

Background: The ascomycete fungi Cylindrocarpon destructans (Cd) and Fusarium solani (Fs) cause ginseng root rot and significantly reduce the quality and yield of ginseng. Cd produces the secondary metabolite radicicol, which targets the molecular chaperone Hsp90. Fs is resistant to radicicol, whereas other fungal genera associated with ginseng disease are sensitive to it. Radicicol resistance mechanisms have not yet been elucidated. Methods: Transcriptome analyses of Fs and Cd mycelia treated with or without radicicol were conducted using RNA-seq. All of the differentially expressed genes (DEGs) were functionally annotated using the Fusarium graminearum transcript database. In addition, deletions of two transporter genes identified by RNA-seq were created to confirm their contributions to radicicol resistance. Results: Treatment with radicicol resulted in upregulation of chitin synthase and cell wall integrity genes in Fs and upregulation of nicotinamide adenine dinucleotide dehydrogenase and sugar transporter genes in Cd. Genes encoding an ATP-binding cassette transporter, an aflatoxin efflux pump, ammonium permease 1 (mep1), and nitrilase were differentially expressed in both Fs and Cd. Among these four genes, only the ABC transporter was upregulated in both Fs and Cd. The aflatoxin efflux pump and mep1 were upregulated in Cd, but downregulated in Fs, whereas nitrilase was downregulated in both Fs and Cd. Conclusion: The transcriptome analyses suggested radicicol resistance pathways, and deletions of the transporter genes indicated that they contribute to radicicol resistance.

• Journal of Microbiology and Biotechnology
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• v.32 no.3
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• pp.287-293
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• 2022

The hydroxylation of methane (CH₄) is crucial to the field of environmental microbiology, owing to the heat capacity of methane, which is much higher than that of carbon dioxide (CO₂). Soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily, is essential for the hydroxylation of specific substrates, including hydroxylase (MMOH), regulatory component (MMOB), and reductase (MMOR). The diiron active site positioned in the MMOH α-subunit is reduced through the interaction of MMOR in the catalytic cycle. The electron transfer pathway, however, is not yet fully understood due to the absence of complex structures with reductases. A type II methanotroph, Methylosinus sporium 5, successfully expressed sMMO and hydroxylase, which were purified for the study of the mechanisms. Studies on the MMOH-MMOB interaction have demonstrated that Tyr76 and Trp78 induce hydrophobic interactions through π-π stacking. Structural analysis and sequencing of the ferredoxin domain in MMOR (MMOR-Fd) suggested that Tyr93 and Tyr95 could be key residues for electron transfer. Mutational studies of these residues have shown that the concentrations of flavin adenine dinucleotide (FAD) and iron ions are changed. The measurements of dissociation constants (K_ds) between hydroxylase and mutated reductases confirmed that the binding affinities were not significantly changed, although the specific enzyme activities were significantly reduced by MMOR-Y93A. This result shows that Tyr93 could be a crucial residue for the electron transfer route at the interface between hydroxylase and reductase.

• Journal of Ginseng Research
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• v.41 no.1
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• pp.23-30
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• 2017

Background: Ginsenoside Rg1 is a class of steroid glycoside and triterpene saponin in Panax ginseng. Many studies suggest that Rg1 suppresses adipocyte differentiation in 3T3-L1. However, the detail molecular mechanism of Rg1 on adipogenesis in 3T3-L1 is still not fully understood. Methods: 3T3-L1 preadipocyte was used to evaluate the effect of Rg1 on adipocyte development in the differentiation in a stage-dependent manner in vitro. Oil Red O staining and Nile red staining were conducted to measure intracellular lipid accumulation and superoxide production, respectively. We analyzed the protein expression using Western blot in vitro. The zebrafish model was used to investigate whether Rg1 suppresses the early stage of fat accumulation in vivo. Results: Rg1 decreased lipid accumulation in early-stage differentiation of 3T3-L1 compared with intermediate and later stages of adipocyte differentiation. Rg1 dramatically increased CAAT/enhancer binding protein (C/EBP) homologous protein-10 (CHOP10) and subsequently reduced the $C/EBP{\beta}$ transcriptional activity that prohibited the initiation of adipogenic marker expression as well as triglyceride synthase. Rg1 decreased the expression of extracellular signal-regulated kinase 1/2 and glycogen synthase kinase $3{\beta}$, which are also essential for stimulating the expression of $CEBP{\beta}$. Rg1 also reduced reactive oxygen species production because of the downregulated protein level of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 4 (NOX4). While Rg1 increased the endogenous antioxidant enzymes, it also dramatically decreased the accumulation of lipid and triglyceride in high fat diet-induced obese zebrafish. Conclusion: We demonstrated that Rg1 suppresses early-stage differentiation via the activation of CHOP10 and attenuates fat accumulation in vivo. These results indicate that Rg1 might have the potential to reduce body fat accumulation in the early stage of obesity.

• Journal of Pharmacopuncture
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• v.18 no.2
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• pp.7-18
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• 2015

Objectives: Lawsone (1,4 naphthoquinone) is a non redox cycling compound that can be catalyzed by DT diaphorase (DTD) into 1,2,4-trihydroxynaphthalene (THN), which can generate reactive oxygen species by auto oxidation. The purpose of this study was to evaluate the toxicity of the phytomarker 1,4 naphthoquinone and its metabolite THN by using the molecular docking program AutoDock 4. Methods: The 3D structure of ligands such as hydrogen peroxide ($H_2O_2$), nitric oxide synthase (NOS), catalase (CAT), glutathione (GSH), glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PDH) and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) were drawn using hyperchem drawing tools and minimizing the energy of all pdb files with the help of hyperchem by $MM^+$ followed by a semi-empirical (PM3) method. The docking process was studied with ligand molecules to identify suitable dockings at protein binding sites through annealing and genetic simulation algorithms. The program auto dock tools (ADT) was released as an extension suite to the python molecular viewer used to prepare proteins and ligands. Grids centered on active sites were obtained with spacings of $54{\times}55{\times}56$, and a grid spacing of 0.503 was calculated. Comparisons of Global and Local Search Methods in Drug Docking were adopted to determine parameters; a maximum number of 250,000 energy evaluations, a maximum number of generations of 27,000, and mutation and crossover rates of 0.02 and 0.8 were used. The number of docking runs was set to 10. Results: Lawsone and THN can be considered to efficiently bind with NOS, CAT, GSH, GR, G6PDH and NADPH, which has been confirmed through hydrogen bond affinity with the respective amino acids. Conclusion: Naphthoquinone derivatives of lawsone, which can be metabolized into THN by a catalyst DTD, were examined. Lawsone and THN were found to be identically potent molecules for their affinities for selected proteins.