• Journal of Microbiology and Biotechnology
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• v.27 no.2
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• pp.357-364
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• 2017

In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, $4{\times}FLAG$, or $6{\times}HA$, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, using $Cac1-6{\times}HA$ and $Aca1-4{\times}FLAG$ tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using $aca1{\Delta}$::ACA1-GFP and $aca1{\Delta}$::ACA1-GFP $cac1{\Delta}$ strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.

• The Journal of Internal Korean Medicine
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• v.38 no.3
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• pp.367-375
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• 2017

Objective: To investigate the effect of Artemisiae Iwayomogii Herba, Curcumae Radix, and Aurantii Fructus Immaturus complex extract (ACA) on dyslipidemia-related factor expression and anti-oxidation in HepG2 cells. Method: After treatment with ACA in the HepG2 cells, DPPH, ABTS radical scavenging activity, ROS production, and glutathione (GSH) production were measured. The free fatty acid, lipid peroxidation (MDA), ACAT1, and HMG-CoA reductase mRNA expression were measured in the HepG2 cells after treatment with ACA. Results: 1. DPPH, ABTS radical scavenging activity increased in an ACA concentration-dependent manner. 2. ACA significantly decreased ROS production in comparison to the control group. 3. ACA significantly increased glutathione production. 4. ACA significantly decreased free fatty acid and lipid peroxidation (MDA) in the HepG2 cells. 5. ACA decreased the mRNA expression of ACAT1 and HMG-CoA reductase. Conclusion: These results suggest that Artemisiae Iwayomogii Herba, Curcumae Radix, and Aurantii Fructus Immaturus complex extract (ACA) inhibits dyslipidemia-related factor expression and that it is effective in anti-oxidation. A future in vivo experiment with ACA is needed to investigate the effect on anti-dyslipidemia. It is expected that ACA is effective in anti-dyslipidemia and applied to cardiovascular disease, ischemic heart disease, stroke, etc.

• Microbiology and Biotechnology Letters
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• v.27 no.2
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• pp.96-101
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• 1999

Various side-chains are introduced to the 7-amino position of 7-aminocepha-losporanic acid (7-ACA) to make semi-synthetic cephalosporin antibiotics. In order to convert cephalosporin C (CPC) to 7-ACA, two enzymatic reactions are generally imployed. Glutary1-7-aminocephalosporanic acid (Gl-7-ACA) acylase is involved in the second step where the reaction intermediate, Gl-7-ACa is converted into 7-ACA. It was recently reported that CPC amidase can convert CPC directly into 7-ACA in a single enzymatic reaction. A study was undertaken to screen microorganisms conferring enzyme activity to convert Gl-7-ACA or CPC into 7-ACA by one or two enzymatic reactions. In order to screen the microorganisms rapidly, a non-$\beta$-lactam model compund, glutaryl-$\rho$-nitroanilide, was utilized in an early stage, thereafter the selected microorganisms were examined with real substrates. One microorganism exhibiting both Gl-7-ACA acylase and CPC amidase activities was obtained by the colorimetry method and HPLC assay, and was identified as a strain of Serratia species, designated as Serratia sp. N14.4. The optimal fermentation conditions for Serratia sp. N14.4 was pH9.0 and 3$0^{\circ}C$.

• Parasites, Hosts and Diseases
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• v.61 no.4
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• pp.397-404
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• 2023

Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.

• Journal of Practical Agriculture & Fisheries Research
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• v.14 no.1
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• pp.47-59
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• 2012

The effects of organic calcium compounds (ACa) tree-spray for late growing stage on the mineral nutrition concentration of fruit, russet and quality in 'Fuji' apple were investigated. Optimum spray concentration of leaves by ACa tree-spray during late growth stage were ranged 125 times among 1,000 times. Also, Optimum concentration of fruits was effected to 125 times. Optimum spray times of leaves effected by ACa 500 times tree-spray during late growth stage were effective in one times (Sept. 25) and three times (Sept. 25, Oct. 5, 15). Also, Optimum times concentration of fruit skin was effected to one times (Sept. 25) and fruit flesh was effected two times (Sept. 25, Oct. 5) and three times (Sept. 25, Oct. 5, 15). Phytotoxicity by tree-spray of ACa concentration and times different not occur to leaf and fruit.

• Korean Journal of Poultry Science
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• v.31 no.2
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• pp.73-78
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• 2004

The effect of supplemental yellow ${\beta}$-8-Apo-carotenoic acid ethyl ester (ACA) (0~300mg/kg feed) on its accumulation in each body part of laying hens and the pigmentation in egg yolk was determined. The chickens used in this study were 78-wk-old ISA brown laying hens. ACA significantly affected the color of the bodies of laying hens. The yellowness ($b^*$) but not the redness ($a^*$) and, the lightness ($L^*$) of the skin, and muscle as measured by a colorimeter significantly (P < 0.05) increased when ACA was fed at 50~100 mg/kg feed. Egg yolk color was also significantly affected by feeding ACA longer than 2 days (P < 0.05) at the levels higher than 50 mg/kg feed. Conclusively, ACA is recommended for improving yellowness of skin.

• Molecules and Cells
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• v.40 no.4
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• pp.291-298
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• 2017

MicroRNAs (miRNAs) are short non-coding RNAs that regulate genes posttranscriptionally. Past studies have reported that miR-210 is up-regulated in many cancers including cervical cancer, and plays a pleiotropic role in carcinogenesis. However, its role in regulating response towards anti-cancer agents has not been fully elucidated. We have previously reported that the natural compound 1'S-1'-acetoxychavicol acetate (ACA) is able to induce cytotoxicity in various cancer cells including cervical cancer cells. Hence, this study aims to investigate the mechanistic role of miR-210 in regulating response towards ACA in cervical cancer cells. In the present study, we found that ACA down-regulated miR-210 expression in cervical cancer cells, and suppression of miR-210 expression enhanced sensitivity towards ACA by inhibiting cell proliferation and promoting apoptosis. Western blot analysis showed increased expression of mothers against decapentaplegic homolog 4 (SMAD4), which was predicted as a target of miR-210 by target prediction programs, following treatment with ACA. Luciferase reporter assay confirmed that miR-210 binds to sequences in 3'UTR of SMAD4. Furthermore, decreased in SMAD4 protein expression was observed when miR-210 was overexpressed. Conversely, SMAD4 protein expression increased when miR-210 expression was suppressed. Lastly, we demonstrated that overexpression of SMAD4 augmented the anti-proliferative and apoptosis-inducing effects of ACA. Taken together, our results demonstrated that down-regulation of miR-210 conferred sensitivity towards ACA in cervical cancer cells by targeting SMAD4. These findings suggest that combination of miRNAs and natural compounds could provide new strategies in treating cervical cancer.

• Journal of Integrative Natural Science
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• v.9 no.3
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• pp.171-176
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• 2016

Rap1 is a key regulator of cell adhesion and migration. Although increasing evidence indicates that the Rap1 signaling pathway is involved in the process of bone remodeling, the mechanism by which Rap1 regulates osteoblastic differentiation and cell adhesion remains unknown. Here, we investigated the morphological characteristics and osteoblastic differentiation of cells expressing constitutively activated form of Rap1A (Rap1ACA) or Rap1 GTPase activating protein Rap1GAP and found that activated Rap1 induces osteoblastic differentiation and cell adhesion as well as cell spreading. When osteoblastic differentiation was induced, Rap1ACA cells showed considerably higher levels of calcium deposits than the wild-type and Rap1GAP-overexpressing cells did. Rap1ACA cells showed increased spreading and size, as well as strong cell adhesion and significantly decreased growth rates. F-actin staining using phalloidin revealed several thin thread-like filopodia around the protrusions in Rap1ACA cells, which possibly contribute to the increased cell adhesion.

• Journal of the Microelectronics and Packaging Society
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• v.12 no.1 s.34
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• pp.9-16
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• 2005

This paper presents the development of new anisotropic conductive adhesives with enhanced thermal conductivity for the wide use of adhesive flip chip technology with improved reliability under high current density condition. The continuing downscaling of structural profiles and increase in inter-connection density in flip chip packaging using ACAs has given rise to reliability problem under high current density. In detail, as the bump size is reduced, the current density through bump is also increased. This increased current density also causes new failure mechanism such as interface degradation due to inter-metallic compound formation and adhesive swelling due to high current stressing, especially in high current density interconnection, in which high junction temperature enhances such failure mechanism. Therefore, it is necessary for the ACA to become thermal transfer medium to improve the lifetime of ACA flip chip joint under high current stressing condition. We developed thermally conductive ACA of 0.63 W/m$\cdot$K thermal conductivity using the formulation incorporating $5 {\mu}m$ Ni and $0.2{\mu}m$ SiC-filled epoxy-bated binder system to achieve acceptable viscosity, curing property, and other thermo-mechanical properties such as low CTE and high modulus. The current carrying capability of ACA flip chip joints was improved up to 6.7 A by use of thermally conductive ACA compared to conventional ACA. Electrical reliability of thermally conductive ACA flip chip joint under current stressing condition was also improved showing stable electrical conductivity of flip chip joints. The high current carrying capability and improved electrical reliability of thermally conductive ACA flip chip joint under current stressing test is mainly due to the effective heat dissipation by thermally conductive adhesive around Au stud bumps/ACA/PCB pads structure.

• Journal of Microbiology
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• v.37 no.4
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• pp.200-205
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• 1999

7-Aminocephalosporanic acid (7-ACA) is the initial compound in preparation of cephalosporin antibiotics widely used in clinical treatment. Bacteria producing glutaryl 7-ACA acylase, which convert cephalosporin C to 7-ACA, has been screened in soil samples. A bacterial strain exhibiting high glutaryl 7-ACA acylase activity, designated KAC-1, was isolated and identified as a strain of Pseudomonas diminuta by characterizing its morphological and physiological properties. The screening procedures include culturing on enrichment media containing glutaric acid, glutamate, and glutaryl 7-aminocephalosporanic acid as selective carbon sources. To enhance enzyme production, optimal cultivation conditions were investigated. This strain grew optimally at pH 7 to 9 and in temperatures of 20 to 40 C, but acylase production was higher when the strain was grown at 25 C. Glutaric acid, glutamate and glucos also acted as inducers for acylase production. In a jar fermenter culture, P. diminuta KAC-1 produce acylase in a growth-associated manner. The substrate specificity of KAC-1 acylase by cell extract showed that this enzyme had specificity toward glutaryl 7-ACA, glutaryl 7-ADCA, but not cephalosporin C.