• The Plant Pathology Journal
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• v.32 no.5
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• pp.452-459
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• 2016

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-${\beta}$-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

• Journal of Microbiology
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• v.45 no.2
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• pp.179-184
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• 2007

Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.

• Korean Journal of Plant Resources
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• v.23 no.3
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• pp.207-213
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• 2010

Hovenia dulcis Thunb fruits were successively extracted with hot water, water, methanol, ethyl acetate, and chloroform. The crude extracts were investigated for potential antioxidant by measuring scavenging against DPPH free radicals, reducing power, superoxide radicals, and protection of protein damage and cultured cells from a lethal dose of hydrogen peroxide ($H_2O_2$). In all chemical assays used, the hot water extract of H. dulcis fruits, which contained $61.14{\pm}2.57$ (Tannic acid mg/g extract, n=3) of total phenolic compounds contents exhibited highest activity in in vitro models of DPPH free radical scavenging activity, reducing power assay, superoxide radical scavenging activity and protection of protein damage. In addition, the hot water extract protected cultured RAW 264.7 macrophages from a lethal dose of $H_2O_2$ and reduced reactive oxygen species level in RAW 264.7 cells.

• The Plant Pathology Journal
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• v.17 no.6
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• pp.388.4-389
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• 2001

• The Plant Pathology Journal
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• v.30 no.2
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• pp.151-158
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• 2014

Three double-stranded RNAs (dsRNAs), approximately 1.85, 1.65 and 1.27 kb in size, were detected in an isolate of Cytospora sacchari from Iran. Partial nucleotide sequence revealed a 1,284 bp segment containing one ORF that potentially encodes a 405 aa protein. This protein contains conserved motifs related to RNA dependent RNA polymerases (RdRp) that showed similarity to RdRps of partitiviruses. The results indicate that these dsRNAs represent a novel Partitivirus that we tentatively designate Cytospora sacchari partitivirus (CsPV). Treatment of the fungal strain by cyclohexamide and also hyphal tip culture had no effect on removing the putative virus. Phylogenetic analysis of putative RdRp of CsPV and other partitiviruses places CsPV as a member of the genus Partitivirus in the family Partitiviridae, and clustering with Aspergillus ochraceous virus 1.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1311-1319
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• 2016

Alternaria brassicicola (Schwein.) invades Brassicaceae and causes black spot disease, significantly lowering productivity. Mitogen-activated protein kinases (MAPKs) and their upstream kinases, including MAPK kinases (MAPKKs) and MAPKK kinases (MAPKKK), comprise one of the most important signaling pathways determining the pathogenicity of diverse plant pathogens. The AbSte7 gene in the genome of A. brassicicola was predicted to be a homolog of yeast Ste7, a MAPKK; therefore, the function was characterized by generating null mutant strains with a gene replacement method. AbSte7 replacement mutants (RMs) had a slower growth rate and altered colony morphology compared with the wild-type strain. Disruption of the AbSte7 gene resulted in defects in conidiation and melanin accumulation. AbSte7 was also involved in the resistance pathways in salt and oxidative stress, working to negatively regulate salt tolerance and positively regulate oxidative stress. Pathogenicity assays revealed that AbSte7 RMs could not infect intact cabbage leaves, but only formed very small lesions in wounded leaves, whereas typical lesions appeared on both intact and wounded leaves inoculated with the wild-type strain. As the first studied MAPKK in A. brassicicola, these data strongly suggest that the AbSte7 gene is an essential element for the growth, development, and pathogenicity of A. brassicicola.

• Molecules and Cells
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• v.45 no.3
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• pp.148-157
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• 2022

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.

• BMB Reports
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• v.31 no.6
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• pp.572-577
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• 1998

A soluble protein from Saccharomyces cerevisiae provides protection against a thiol-containing oxidation system but not against an oxidation system without thiol. This 25-kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was thus named thioredoxin peroxidase. The protective role of thioredoxin peroxidase against ionizing radiation, which generates reactive oxygen species harmful tocellular function, was investigated in wild-type and mutant yeast strains in which the tsa gene encoding thioredoxin peroxidase was disrupted by homologous recombination. Upon exposure to ionizing radiation, there was a distinct difference between these two strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein. Activities of other antioxidant enzymes, such as catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase were increased at 200-600 Gy of irradiation in wild-type cells. However, the activities of antioxidant enzymes were not significantly changed by ionizing radiation in thioredoxin peroxidase-deficient mutant cells. These results suggest that thioredoxin peroxidase acts as an antioxidant enzyme in cellular defense against ionizing radiation through the removal of reactive oxygen species as well as in the protection of antioxidant enzymes.

• The Plant Pathology Journal
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• v.34 no.5
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• pp.426-434
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• 2018

During the spring season of 2014, a total of 148 melon and watermelon leaf samples were collected from symptomatic and asymptomatic plants in the western and southwestern regions of Saudi Arabia and were tested for the presence of Watermelon chlorotic stunt virus (WmCSV) and other suspected cucurbit viruses by double antibody sandwich enzyme-linked immunosorbent assays. Ninety-eight samples were found to be positive for the presence of WmCSV, nine samples were positive for the presence of Cucurbit yellow stunting disorder virus (CYSDV), and 22 showed a mixed infection with both WmCSV and CYSDV. No other cucurbit viruses were detected in any of the samples. Host range experiments revealed that eight out of fourteen tested plant species were susceptible to WmCSV. PCR products of approximately 1.2 kb were obtained after amplification using primers specifically targeting the coat protein region of WmCSV. Positive PCR results were confirmed by dot blot hybridization. Coat protein gene sequences from eleven WmCSV isolates indicated that the highest identity was between the 104WMA-SA isolate from the Wadi Baish location and a previously reported isolate from the AL-Lith location in Saudi Arabia. The lowest identity was observed between the 42WMA-SA isolate and an isolate from Palestine.

• Journal of Microbiology and Biotechnology
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• v.17 no.9
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• pp.1452-1459
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• 2007

The Dps protein, which is overexpressed in harsh environments, is known to playa critical role in the protection of DNA against oxidative stresses. In this study, the roles of Fur in the expression of the dps gene in Salmonella and the protection mechanisms against oxidative stress in Salmonella cells preexposed to iron-stress were investigated. Two putative Fur boxes were predicted within the promoter region of the S. typhimurium dps gene. The profile of dps expression performed by the LacZ reporter assay revealed growth-phase dependency regardless of iron-status under the culture conditions. The fur mutant, $_X4659$, evidenced a reduced level of ${\beta}$-galactosidase as compared to the wild-type strain. The results observed after the measurement of the Dps protein in various Salmonella regulatory mutants were consistent with the results acquired in the reporter assay. This evidence suggested that Fur performs a function as a subsidiary regulator in the expression of dps. The survival ability of Salmonella strains after exposure to oxidative stress demonstrated that the Dps protein performs a pivotal function in the survival of stationary-phase S. typhimurium against oxidative stress. Salmonella cells grown in iron-restricted condition required Dps for full protection against oxidative stress. The CK24 (${\Delta}dps$) cells grown in iron-replete condition survived at a rate similar to that observed in the wild-type strain, thereby suggesting the induction of an unknown protection mechanism(s) other than Dps in this condition.