[Show abstract][Hide abstract] ABSTRACT: The hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse genetic approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, on the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse genetic studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication.
[Show abstract][Hide abstract] ABSTRACT: Various studies have been conducted to investigate effects of dams on river ecosystems, but less information is available regarding damming impacts on downstream denitrification. We measured denitrification enzyme activity (potential denitrification rate) and denitrifier abundances (using nirS, nirK, and nosZ as markers) in dammed headstreams of the Nakdong River in South Korea. Sediments in Phragmites-dominated riparian areas and in-stream areas across streams (dammed vs. reference) with different streambed materials (gravel and sand) were sampled occasionally. We hypothesized that (i) the higher available N and C contents in sediments downstream of dams foster larger denitrifier communities than in the reference system and (ii) differences in potential denitrification rates across the systems correspond with denitrifier abundances. Despite 30 years of different hydrological management with dams and greater inorganic N and DOC contents in sediments downstream of dams, compared to the references, abundances of denitrifier communities and potential denitrification rates within the whole sediment were not significantly different across the systems. However, nirS and nosZ denitrifier abundances and potential denitrification rates were considerably increased in specific sediments downstream of dams (gravelly riparian and sandy in-stream) with regard to flooding events and seasonal temperature variation. nirK was not amplified in all sediments. Canonical correspondence analyses (CCA) revealed that the relationship between abundances of denitrifier communities and nutrient availabilities and potential denitrification rates was a weak one.
Korean Journal of Microbiology 06/2014; 50(2):137-151.
[Show abstract][Hide abstract] ABSTRACT: Poly-γ-glutamic acid (γ-PGA) is an anionic polypeptide secreted by Bacillus sp. that has been shown to activate immune cells through interactions with toll-like receptor 4 (TLR4). However, its ability to induce the type I interferon (IFN) response has not yet been characterized. Here, we demonstrate that γ-PGA induces type I IFN signaling pathway via the TLR4 signaling pathway. The induction required both myeloid differentiation factor 2 (MD2) and the pattern-recognition receptor CD14, which are two TLR4-associated accessory proteins. The γ-PGA with high molecular weights (2000 and 5000 kDa) was able to activate the subsequent signals through TLR4/MD2 to result in dimerization of IRF-3, a transcription factor required for IFN gene expression, leading to increases in mRNA levels of the type I IFN-response genes, 2'-5' OAS and ISG56. Moreover, γ-PGA (2000 kDa) displayed an antiviral activity against SARS coronavirus and hepatitis C virus. Our results identify high-molecular weight γ-PGA as a TLR4 ligand and demonstrate that γ-PGA requires both CD14 and MD2 for the activation of type I IFN responses. Our results suggest that the microbial biopolymer γ-PGA may have therapeutic potential against a broad range of viruses sensitive to type I IFNs.
[Show abstract][Hide abstract] ABSTRACT: During the past several decades, the Taguchi robust design method has been widely used in various fields successfully. On the other hand, some researchers and practitioners have criticized the method with respect to the way of utilizing orthogonal arrays, the signal-to-noise ratio as a performance measure, data analysis methods, etc., and proposed alternative approaches to robust design. This paper introduces the Taguchi method first, evaluates the validity of the criticisms, and discusses advantages and disadvantages of each alternative. Finally, research issues to be addressed for effective robust design are presented.
Journal of Korean Institute of Industrial Engineers. 01/2013; 39(5).
[Show abstract][Hide abstract] ABSTRACT: Tundra ecosystem is of importance for its high accumulation of organic carbon and vulnerability to future climate change. Microorganisms play a key role in carbon dynamics of the tundra ecosystem by mineralizing organic carbon. We assessed both ecosystem process rates and community structure of Bacteria, Archaea, and Fungi in different soil layers (surface organic layer and subsurface mineral soil) in an Arctic soil ecosystem located at Spitsbergen, Svalbard during the summer of 2008 by using biochemical and molecular analyses, such as enzymatic assay, terminal restriction fragment length polymorphism (T-RFLP), quantitative polymerase chain reaction (qPCR), and pyrosequencing. Activity of hydrolytic enzymes showed difference according to soil type. For all three microbial communities, the average gene copy number did not significantly differ between soil types. However, archaeal diversities appeared to differ according to soil type, whereas bacterial and fungal diversity indices did not show any variation. Correlation analysis between biogeochemical and microbial parameters exhibited a discriminating pattern according to microbial or soil types. Analysis of the microbial community structure showed that bacterial and archaeal communities have different profiles with unique phylotypes in terms of soil types. Water content and hydrolytic enzymes were found to be related with the structure of bacterial and archaeal communities, whereas soil organic matter (SOM) and total organic carbon (TOC) were related with bacterial communities. The overall results of this study indicate that microbial enzyme activity were generally higher in the organic layer than in mineral soils and that bacterial and archaeal communities differed between the organic layer and mineral soils in the Arctic region. Compared to mineral soil, peat-covered organic layer may represent a hotspot for secondary productivity and nutrient cycling in this ecosystem.
[Show abstract][Hide abstract] ABSTRACT: Heat shock protein 90 (Hsp90), which chaperones multiple client proteins, has been shown to be implicated in HCV replication. Pharmacological inhibitors of Hsp90 display an anti-HCV activity. However, little is known about the mechanisms of regulation of HCV replication by Hsp90. Here, we show that Hsp90 inhibition by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) destabilizes phosphoinositide-dependent kinase-1 (PDK1), an upstream kinase of the protein kinase C-related kinase 2 (PRK2) responsible for phosphorylation of HCV RNA polymerase, through the proteosome pathway. Destabilization of PDK1 led to inhibition of phosphorylation of the viral RNA polymerase through a decrease in the abundance of active form PRK2 level. Consequently, Hsp90 inhibition resulted in suppression of HCV replication both in human hepatoma Huh7 cells harboring an HCV subgenomic replicon and in HCV-infected cells. 17-DMAG treatment did not interfere with HCV internal ribosome entry site-mediated translation and the cell cycle in Huh7 cells. Co-treatment of 17-DMAG with interferon-α or HA1077, an inhibitor of PRK2, enhanced the anti-HCV activity of 17-DMAG. Taken together, these findings suggest that Hsp90 plays a critical role in the regulation of HCV RNA polymerase phosphorylation via the PDK1-PRK2 signaling pathway.
Biochemical and Biophysical Research Communications 04/2012; 421(1):112-8. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of elevated CO(2) on soil bacterial community with upland vegetation have been widely studied, but limited information is available regarding responses of denitrifier and methanogen communities to elevated CO(2) in wetland ecosystems. Using restriction fragment length polymorphism (RFLP), terminal RFLP analysis, and real-time quantitative PCR, we compared communities of denitrifiers and methanogens in a laboratory-scale wetland system planted with one of three macrophytes, Typha latifolia, Scirpus lacustris, or Juncus effusus, after 110 days of incubation. Our study showed that elevated CO(2) could affect community structures of both denitrifiers and methanogens, each of which had a unique response pattern. In particular, elevated CO(2) shifted nirS-containing community with a unique structure irrespective of vegetation type. mcrA-containing community appeared to shift to community with unique types of hydrogenotrophs under elevated CO(2) conditions. The change of dissolved organic carbon driven by elevated CO(2) appeared to be related with the shift of both denitrifiers and methanogens. Overall, this study indicates that elevated CO(2) could change the community structure of denitrifiers and methanogens temporarily. These results also suggest a presence of stable dominant populations that were not substantially affected by changes in CO(2) concentration.
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) infection causes chronic liver disease and is a major public health problem worldwide. The aim of this study was to evaluate the potential of Monascus pigment derivatives, which were derived from a microbial secondary metabolite synthesized from polyketides by Monascus spp., as HCV antiviral agents.
We performed an in vitro RNA-dependent RNA polymerase (RdRp) assay to screen for HCV RdRp inhibitors. The anti-HCV activity of RdRp inhibitors in HCV-replicating cells was evaluated by quantification of the RNA viral genome. Molecular docking analysis was performed to predict the binding sites of the selected RdRp inhibitors.
We have identified a Monascus pigment and its derivatives as inhibitors of the HCV NS5B RdRp. A group of Monascus orange pigment (MOP) amino acid derivatives, in which the reactive oxygen moiety was changed to amino acids, significantly inhibited HCV replication. Further, combination of the MOP derivatives (Phe, Val or Leu conjugates) with interferon (IFN)-α inhibited HCV replication more than IFN-α treatment alone. Lastly, molecular docking studies indicate the inhibitors may bind to a thumb subdomain allosteric site of NS5B. The antiviral activity of the MOP derivatives was related to a modulation of the mevalonate pathway, since the mevalonate-induced increase in HCV replication was suppressed by the MOP compounds.
Our results identify amino acid derivatives of MOP as potential anti-HCV agents and suggest that their combination with IFN-α might offer an alternative strategy for the control of HCV replication.
Journal of Antimicrobial Chemotherapy 11/2011; 67(1):49-58. · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Constructed wetlands are generally created for water quality amelioration using natural biogeochemical processes including denitrification. To have maximal denitrification rates in constructed wetlands, both optimal environmental conditions and activation of denitrifiers are required. In this study, we monitored temporal dynamics of the community structure of denitrifying bacteria by terminal restriction fragment length polymorphism (T-RFLP) in newly constructed wetlands over 2 growing seasons. We also measured denitrification rates and denitrifying enzyme activity (DEA) using an acetylene blocking method. Community structure of denitrifying bacteria differed between 2 years, and dominant T-RFs changed over the period. In addition, the composition of denitrifying bacteria became simplified in the second year. Along with changes in community structure of denitrifying bacteria, DEA increased substantially in the second year compared with that in the first year. This result suggests that several dominant and active denitrifiers may induce higher potential for denitrification as wetlands became stabilized. However, actual denitrification rates did not exhibit an annual difference, but were more strongly influenced by environmental conditions such as temperature, pH, and DOC concentrations. Overall results of this study indicate that active denitrifiers may dominate and denitrification potentials increase as a newly constructed wetland becomes stabilized, but actual denitrification rates are mainly constrained by environmental conditions such as temperature, pH and carbon availability.
European Journal of Soil Biology - EUR J SOIL BIOL. 01/2011; 47(1):24-29.
[Show abstract][Hide abstract] ABSTRACT: We assessed the temporal dynamics of bacterial and fungal communities in a soil ecosystem supporting genetically modified (GM) rice (Oryza sativa L., ABC-TPSP; fusion of trehalose-6-phosphate synthase and phosphatase). Using terminal restriction fragment length polymorphism analysis and real-time quantitative PCR, we compared bacterial and fungal communities in the soils underlying GM rice (ABC-TPSP), and its host cultivar (Nakdong) during growing seasons and non-growing seasons. Overall, the soils supporting GM and non-GM rice did not differ significantly in diversity indices, including ribotype numbers, for either bacteria or fungi. The diversity index (H) in both the bacterial and fungal communities was correlated with water content, dissolved organic carbon (DOC), and ammonium nitrogen, and the correlation was stronger in fungi than in bacteria. Multivariate analysis showed no differences in microbial community structures between the two crop genotypes, but such differences did appear in time, with significant changes observed after harvest. Gene copy number was estimated as 10(8)~10(11) and 10(5)~10(7) per gram of soil for bacteria and fungi, respectively. As observed for community structure, the rice genotypes did not differ significantly in either bacterial- or fungal-specific gene copy numbers, although we observed a seasonal change in number. We summarize the results of this study as follows. (1) GM rice did not influence soil bacterial and fungal community structures as compared to non-GM rice in our system, (2) both bacterial and fungal communities changed with the growth stage of either rice genotype, (3) fungal communities were less variable than bacterial communities, and (4) although several environmental factors, including ammonium nitrogen and DOC correlated with shifts in microbial community structure, no single factor stood out.
[Show abstract][Hide abstract] ABSTRACT: Hydrologic pulsing, including water level drawdown and subsequent flooding, may have a considerable impact on both biogeochemical processes and microbial communities in wetlands. Since denitrifying bacteria play a key role in water quality improvement in wetlands, changes in their activities and communities with hydrologic pulsing are an important issue. We investigated the responses of in situ denitrification rates, denitrifying bacterial community structure and their quantities using nitrite reductase (nir) S gene under different hydrological pulsing conditions in created wetlands in central Ohio USA. Average denitrification rates, measured from 4 different sampling locations, were 302, 133, 71 and 271 μg N2O–N m−2 h−1 during inundated, saturated, drying and reflooding periods, respectively. In particular, the denitrification rates in shallow water level marsh areas (SM) followed by deepwater level marsh areas (DM) showed more sensitivity and magnitude of changes to hydrologic pulsing events than did non-vegetated deepwater areas. This may have been due to the high aerobic decomposition during the drying period and nutrient flushing in shallower marsh areas after the reflooding event. In contrast, the community structure and diversity of denitrifiers based on terminal-restricted fragment length polymorphism (T-RFLP) analysis showed no significant change due to hydrologic pulsing. Instead, the presence and absence of vegetation altered denitrifying bacterial community structure. The nirS gene copy number remained relatively constant with only minor increases during water level drawdown followed by a significant decrease when a sudden reflooding event occurred. These results indicate that environmental disturbances, such as hydrologic pulsing, have a major impact on the denitrification process, but less impact on the community structures of the denitrifying bacteria. In addition, there was no relationship among the denitrification rate, the community structure, and the quantity of denitrifiers, suggesting that changes in denitrification rates during hydrologic pulsing events were not caused by the changes in microbial community structure but more by physicochemical factors, such as substrate availability and hydrology.
[Show abstract][Hide abstract] ABSTRACT: This study explores the characteristics of bacterial and fungal communities of total suspended particles (TSP) in the atmosphere by using various molecular methods. TSP samples were collected on a glass fiber filter at an urban location in the middle of the Korean Peninsula (Seoul) between middle autumn and early winter in 2007. From the aerosol samples, DNA could be extracted and DNA sequences were determined for bacteria and fungi. Terminal restriction length polymorphism (T-RFLP) analysis was applied to analyze the community structure of them. To estimate the concentration of DNA originating from bacterial and fungal communities, we used the quantitative real-time polymerase chain reaction (Q-PCR). Sequence analyses were also used to determine the identity of biological organisms. The number of bacteria and fungi in the air were between 5.19x10(1) and 4.31x10(3) cellsm(-3) and from 9.56x10(1) to 4.22x10(4) cellsm(-3), respectively and bacterium/fungus ratios ranged from 0.09 to 0.76 across the seven sampling dates. Most of the bacterial sequences found in our TSP samples were from Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The fungal sequences were characteristic for Ascomycota, Basidiomycota, and Glomeromycota which are known to actively discharge spores into the atmosphere. The plant sequences could be also detected. We found that large shifts in the community structure of bacteria and fungi were present in our TSP samples collected on different dates. The results demonstrated that in our TSP samples collected at the urban site; (1) there were very diverse bacterial and fungal groups including potential pathogens and allergens and (2) there were temporal shifts in both bacterial and fungal communities in terms of both diversity and abundances across an inter-seasonal period.
Science of The Total Environment 11/2009; 408(6):1349-57. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Decomposition of recalcitrant materials such as phenolics is known to play a pivotal role in organic matter decomposition and nutrient cycling in estuaries. The specific goals of this study were to determine temporal and spatial variations of phenol oxidase and phenolics in estuarine soils, and to elucidate controlling factors for phenol oxidase activity. To achieve these goals, phenol oxidase activity and phenolic content were measured in soils developed along the side of an estuary in the Han River, Korea. Soil samples were collected in three locations with different vegetation: mud flats, Zizania-dominated soils, and Salix-dominated soils. Monthly measurements were also made in a Zizania-dominated site over a year period. Phenol oxidase activity varied between 0.00 and 0.28 diqc min g, whilst phenolic content ranged from 0.0–10.5 μg g. A correlation analysis revealed that phenol oxidase activity exhibited positive correlations with phenolic content in both seasonal and spatial data. The same relationship was found when the data were analysed separately for each site. Unlike peatlands or upland forest soils where negative correlations were often found between phenol oxidase activity and phenolics, substrate induction appears to account for the positive correlation in the present study.
Chemistry and Ecology 01/2009; 25(2):99-106. · 1.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The frequency of drought is anticipated to increase in wetland ecosystems as global warming intensifies. However, information on microbial communities involved in greenhouse gas emissions and their responses to drought remains sparse. We compared the gene abundance of eubacterial 16S rRNA, nitrite reductase (nirS) and methyl coenzyme M reductase (mcrA), and the diversity and composition of eubacteria, methanogens and denitrifiers among bog, fen and riparian wetlands. The gene abundance, diversity and composition significantly differed among wetlands (p < 0.01) with the exception of the diversity of methanogens. The gene abundance was ranked in the order of the bog = fen > riparian wetland, whereas the diversity was in the riparian wetland ≥ fen > bog. In addition, we conducted a short-term drought experiment and compared microbial communities between control (water-logged) and drought (−15 cm) treatments. Drought led to significant decline in the gene abundance in the bog (16S rRNA, nirS, mcrA) (p < 0.01) and fen (16S rRNA, nirS) (p < 0.05), but not in the riparian wetland. There were no differences in the diversity and composition of denitrifiers and methanogens at all sites following drought. Our results imply that denitrifiers and methanogens inhabiting bogs and fens would suffer from short-term droughts, but remain unchanged in riparian wetlands.
Soil Biology and Biochemistry 01/2008; 40(11):2874-2880. · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Effects of elevated CO(2) on soil microorganisms are known to be mediated by various interactions with plants, for which such effects are relatively poorly documented. In this review, we summarize and synthesize results from studies assessing impacts of elevated CO(2) on soil ecosystems, focusing primarily on plants and a variety the of microbial processes. The processes considered include changes in microbial biomass of C and N, microbial number, respiration rates, organic matter decomposition, soil enzyme activities, microbial community composition, and functional groups of bacteria mediating trace gas emission such as methane and nitrous oxide. Elevated CO(2) in atmosphere may enhance certain microbial processes such as CH(4) emission from wetlands due to enhanced carbon supply from plants. However, responses of extracellular enzyme activities and microbial community structure are still controversy, because interferences with other factors such as the types of plants, nutrient availabilitial in soil, soil types, analysis methods, and types of CO(2) fumigation systems are not fully understood.
The Journal of Microbiology 01/2004; 42(4):267-77. · 1.28 Impact Factor