• Journal of Microbiology and Biotechnology
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• v.26 no.11
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• pp.1881-1890
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• 2016

Manganese superoxide dismutase (MnSOD) is a vital enzyme that protects cells from free radicals through eliminating superoxide radicals ($O^{2-}$). Hirudin, a kind of small active peptide molecule, is one of the strongest anticoagulants that can effectively cure thrombus diseases. In this study, we fused Hirudin to the C terminus of human MnSOD with the GGGGS linker to generate a novel dual-feature fusion protein, denoted as hMnSOD-Hirudin. The hMnSOD-Hirudin gene fragment was cloned into the pET15b (SmaI, CIAP) vector, forming a recombinant pET15b-hMnSOD-Hirudin plasmid, and then was transferred into Escherichia coli strain Rosetta-gami for expression. SDS-PAGE was used to detect the fusion protein, which was expected to be about 30 kDa upon IPTG induction. Furthermore, the hMnSOD-Hirudin protein was heavily detected as a soluble form in the supernatant. The purification rate observed after Ni NTA affinity chromatography was above 95%. The hMnSOD-Hirudin protein yield reached 67.25 mg per liter of bacterial culture. The identity of the purified protein was confirmed by western blotting. The hMnSOD-Hirudin protein activity assay evinced that the antioxidation activity of the hMnSOD-Hirudin protein obtained was $2,444.0{\pm}96.0U/mg$, and the anticoagulant activity of the hMnSOD-Hirudin protein was $599.0{\pm}35.0ATU/mg$. In addition, in vitro bioactivity assay showed that the hMnSOD-Hirudin protein had no or little cytotoxicity in H9c2, HK-2, and H9 (human $CD_4{^+}$, T cell) cell lines. Transwell migration assay and invasion assay showed that the hMnSOD-Hirudin protein could suppress human lung cancer 95-D cell metastasis and invasion in vitro.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.104.1-104
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• 2003

To screen for potential biocontrol agents against postharvest disease of garlics caused by Penicillium hirsutum, a total of 933 isolates (432 fungi and 501 bacteria) were isolated from the rhizoshere or rhizoplane of garlics. Among them, Pantoea agglomerans isolate 59-4 (Pa 59-4) was selected for a potential biocontrol agent by in vivo wounded garlic bulb assay, When the spore suspension (10$\^$5/ spores/$m\ell$) of Penicillium hirsutum was co-inoculated with spore or cell suspension of each fungal or bacterial isolate on wounded garlics, the isolate highly suppressed disease development. Soaking garlic bulbs in the suspension of Pa 59-4 significantly reduced garlic decay from p. hirsutum. However, Pa 59-4 did not inhibit the mycelial growth of P. hirsutum in dual-culture with P. hirsutum on Tryptic soy agar. In order to elucidate mode of action of Pa 59-4 nutrient competition between Pa 59-4 and P. hirsutum was investigated using tissue culture plates with cylinder inserts containing defusing membrane reported by Janisiewicz et al. The results showed that Pa 59-4 effectively suppressed spore germination and mycelial growth of blue mold in the low concentration (0.5％) of garlic juice, but did not suppress those of blue mold in the higher concentration (5％) of garlic juice. This result suggests that the mechanism in biocontrol of garlic blue mold by Pa 59-4 may involve in nutrient competition with P. hirsutum on garlic bulbs.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.5
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• pp.620-631
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• 2015

A total of 59 lactic acid bacteria (LAB) strains were isolated from corn stover silage. According to phenotypic and chemotaxonomic characteristics, 16S ribosomal DNA (rDNA) sequences and recA gene polymerase chain reaction amplification, these LAB isolates were identified as five species: Lactobacillus (L.) plantarum subsp. plantarum, Pediococcus pentosaceus, Enterococcus mundtii, Weissella cibaria and Leuconostoc pseudomesenteroides, respectively. Those strains were also screened for antimicrobial activity using a dual-culture agar plate assay. Based on excluding the effects of organic acids and hydrogen peroxide, two L. plantarum subsp. plantarum strains ZZU 203 and 204, which strongly inhibited Salmonella enterica ATCC $43971^T$, Micrococcus luteus ATCC $4698^T$ and Escherichia coli ATCC $11775^T$ were selected for further research on sensitivity of the antimicrobial substance to heat, pH and protease. Cell-free culture supernatants of the two strains exhibited strong heat stability (60 min at $100^{\circ}C$), but the antimicrobial activity was eliminated after treatment at $121^{\circ}C$ for 15 min. The antimicrobial substance remained active under acidic condition (pH 2.0 to 6.0), but became inactive under neutral and alkaline condition (pH 7.0 to 9.0). In addition, the antimicrobial activities of these two strains decreased remarkably after digestion by protease K. These results preliminarily suggest that the desirable antimicrobial activity of strains ZZU 203 and 204 is the result of the production of a bacteriocin-like substance, and these two strains with antimicrobial activity could be used as silage additives to inhibit proliferation of unwanted microorganism during ensiling and preserve nutrients of silage. The nature of the antimicrobial substances is being investigated in our laboratory.

• Journal of Neurogastroenterology and Motility
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• v.24 no.4
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• pp.656-668
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• 2018

Background/Aims MicroRNAs (miRNAs) were reported to be responsible for intestinal permeability in diarrhea-predominant irritable bowel syndrome (IBS-D) rats in our previous study. However, whether and how miRNAs regulate visceral hypersensitivity in IBS-D remains largely unknown. Methods We established the IBS-D rat model and evaluated it using the nociceptive visceral hypersensitivity test, myeloperoxidase activity assay, restraint stress-induced defecation, and electromyographic (EMG) activity. The distal colon was subjected to miRNA microarray analysis followed by isolation and culture of colonic epithelial cells (CECs). Bioinformatic analysis and further experiments, including dual luciferase assays, quantitative real-time polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay, were used to detect the expression of miRNAs and how it regulates visceral hypersensitivity in IBS-D rats. Results The IBS-D rat model was successfully established. A total of 24 miRNAs were differentially expressed in the distal colon of IBS-D rats; 9 were upregulated and 15 were downregulated. Among them, the most significant upregulation was miR-200a, accompanied by downregulation of cannabinoid receptor 1 (CNR1) and serotonin transporter (SERT). MiR-200a mimic markedly inhibited the expression of CNR1/SERT. Bioinformatic analysis and luciferase assay confirmed that CNR1/SERT are direct targets of miR-200a. Rescue experiments that overexpressed CNR1/SERT significantly abolished the inhibitory effect of miR-200a on the IBS-D rats CECs. Conclusions This study suggests that miR-200a could induce visceral hyperalgesia by targeting the downregulation of CNR1 and SERT, aggravating or leading to the development and progression of IBS-D. MiR-200a may be a regulator of visceral hypersensitivity, which provides potential targets for the treatment of IBS-D.

• Mycobiology
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• v.40 no.4
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• pp.244-251
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• 2012

In vitro and greenhouse screening of seven rhizobacterial isolates, AB05, AB10, AB11, AB12, AB14, AB15 and AB17, was conducted to investigate the plant growth promoting activities and inhibition against anthracnose caused by Colletotrichum acutatum in pepper. According to identification based on 16S rDNA sequencing, the majority of the isolates are members of Bacillus and a single isolate belongs to the genus Paenibacillus. All seven bacterial isolates were capable of inhibiting C. acutatum to various degrees. The results primarily showed that antibiotic substances produced by the selected bacteria were effective and resulted in strong antifungal activity against the fungi. However, isolate AB15 was the most effective bacterial strain, with the potential to suppress more than 50% mycelial growth of C. acutatum in vitro. Moreover, antibiotics from Paenibacillus polymyxa (AB15) and volatile compounds from Bacillus subtilis (AB14) exerted efficient antagonistic activity against the pathogens in a dual culture assay. In vivo suppression activity of selected bacteria was also analyzed in a greenhouse with the reference to their prominent in vitro antagonism efficacy. Induced systemic resistance in pepper against C. acutatum was also observed under greenhouse conditions. Where, isolate AB15 was found to be the most effective bacterial strain at suppressing pepper anthracnose under greenhouse conditions. Moreover, four isolates, AB10, AB12, AB15, and AB17, were identified as the most effective growth promoting bacteria under greenhouse conditions, with AB17 inducing the greatest enhancement of pepper growth.

• Journal of the East Asian Society of Dietary Life
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• v.20 no.1
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• pp.30-36
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• 2010

This study was conducted to investigate the effects of blueberry extract on cell death, ROS and gene expression patterns associated with the anti-cancer activity in human breast cancer MCF7 cells. To accomplish this, 20 mg/mL concentration of blueberry extract was added to the cell culture for 0, 6, 12, 24 or 48 h, after which the effects were evaluated by various analyses. MTT assay showed that the cellular activities decreased rapidly during the first 12 h of treatment. During this period, dual staining with Hoechst33322 and propidium iodide also produced a similar trend in which the dead or dying cells increased sharply. Furthermore, evaluation of BrdU incorporation as an index for cell proliferation revealed a marked decrease during the first 12 h of treatment, suggesting that anticancer activity involves the inhibition of cell proliferation and induces cell death. ROS also increased according to the duration of the treatment, indicating intracellular accumulation is associated with the cell death. RT-PCR analysis revealed significant decreases in anti-apoptotic (Bax) and increases in pro-apoptotic gene expressions (Bci-2, caspase- 3, and 9) (p<0.05). Taken these together, blueberry extract induces ROS accumulation in MCF7 cells, causing inhibition of cell proliferation and eventually leading to cell death. This cell death was associated with apoptotic gene expression in blueberry-treated cells for up to 24 h.

• The Plant Pathology Journal
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• v.35 no.6
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• pp.674-683
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• 2019

Some species of the Trichoderma genus are reported as the major problem in oak wood mushroom production in Korea. In spite of economic loss by the fungi, scientific information on airborne Trichoderma species is not much available. To generate information for disease management development we analyzed airborne Trichoderma. A total of 1,063 fungal isolates were purely obtained from indoor air sampling of cultivation houses used for oak wood mushroom using sawdust media. Among the obtained isolates, 248 isolates were identified as Trichoderma fungi including T. harzianum, T. atroviride, T. citrinoviride, and T. pseudokoningii, by morphological and molecular analysis. T. harzianum was dominant among the four identified species. All the four Trichoderma species grew fast on solid nutrient media tested (potato dextrose agar [PDA], malt extract agar [MEA], Czapek's Dox + yeast extract agar [CYA] and cornmeal dextrose agar). Compact mycelia growth and mass spore production were better on PDA and CYA. In addition, T. harzianum and T. citrinoviride formed greenish and yellowish mycelium and spores on PDA and CYA. Greenish and yellowish pigment was saturated into PDA only by T. pseudokoningii. These four Trichoderma species could produce extracellular enzymes of sawdust substrate degradation such as β-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease. Their mycelia inhibited the growth of oak wood mushroom mycelia of two tested cultivars on dual culture assay. Among of eleven antifungal agents tested, benomyl was the best to inhibit the growth of the four Trichoderma species. Our results demonstrate that the airborne Trichoderma fungi need to be properly managed in the cultivation houses for safe mushroom production.

• The Plant Pathology Journal
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• v.35 no.6
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• pp.598-608
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• 2019

Endophytic fungi have received much attention as plant growth promoters as well as biological control agents against many plant pathogens. In this study, 30 endophytic fungal species, isolated from various plants in China, were evaluated using in vitro dual culture assay against Fusarium oxysporum f. sp. cucumerinum, causing wilt in cucumber. The results of the present study clearly showed that all the 30 endophytic fungal isolates were highly capable of inhibiting the mycelial colony growth of Fusarium oxysporum f. sp. cucumerinum with inhibition % over 66% as compared to control treatments. Among all of them, 5 isolates were highly effective such as, Penicillium sp., Guignardia mangiferae, Hypocrea sp., Neurospora sp., Eupenicillium javanicum, and Lasiodiplodia theobromae, respectively. The Penicillium sp. and Hypocrea sp. were highly effective as compared to other isolates. From in vitro results 10 best isolates were selected for greenhouse studies. The results of the greenhouse studies showed that among all of them 3 endophytic fungal isolates successfully suppressed wilt severity when co-inoculation with pathogen Fusarium. oxysporum f. sp. cucumerinum. The endophytic fungi also enhanced plant growth parameters of the host plants, the antagonistic fungal isolates increased over all plant height, aerial fresh, and dry weight as compared to control.

• Journal of Microbiology and Biotechnology
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• v.31 no.9
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• pp.1241-1255
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• 2021

This study was carried out to explore a non-chemical strategy for enhancing productivity by employing some antagonistic rhizobacteria. One hundred eighteen bacterial isolates were obtained from the rhizospheric zone of various crop fields of Gangwon-do, Korea, and screened for antifungal activity against Fusarium wilt (Fusarium oxysporum f. sp. lactucae) in lettuce crop under in vitro and in vivo conditions. In broth-based dual culture assay, fourteen bacterial isolates showed significant inhibition of mycelial growth of F. oxysporium f. sp. lactucae. All of the antagonistic isolates were further characterized for the antagonistic traits under in vitro conditions. The isolates were identified on the basis of biochemical characteristics and confirmed at their species level by 16S rRNA gene sequencing analysis. Arthrobacter sulfonivorans, Bacillus siamensis, Bacillus amyloliquefaciens, Pseudomonas proteolytica, four Paenibacillus peoriae strains, and Bacillus subtilis were identified from the biochemical characterization and 16S rRNA gene sequencing analysis. The isolates EN21 and EN23 showed significant decrease in disease severity on lettuce compared to infected control and other bacterial treatments under greenhouse conditions. Two bacterial isolates, EN4 and EN21, were evaluated to assess their disease reduction and growth promotion in lettuce in field conditions. The consortium of EN4 and EN21 showed significant enhancement of growth on lettuce by suppressing disease caused by F. oxysporum f. sp. lactucae respectively. This study clearly indicates that the promising isolates, EN4 (P. proteolytica) and EN21 (Bacillus siamensis), can be commercialized and used as biofertilizer and/or biopesticide for sustainable crop production.

• Research in Plant Disease
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• v.19 no.1
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• pp.12-20
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• 2013

To isolate antagonistic bacteria against sclerotinia rot of lettuce, caused by Sclerotinia sclerotiorum, soil samples were collected from the diseased greenhouse field in Namyangju city, Gyeong-gi province from 2007 to 2008. A total of 196 bacterial isolates were isolated using serial dilution method. In dual culture assay in vitro, 26 isolates showed more than 80% of inhibition rates of mycelial growth of S. sclerotiorum. Based on 16S rDNA sequence analysis, the 26 isolates were identified as Bacillus megaterium, B. cereus, B. subtilis, Arthrobacter nicotianae, A. ramosus, Pseudomonas filiscindens, Stenotrophomonas maltophilia, Brevibacterium frigoritolerans and Sphingobacterium faecium. The 26 isolates inhibited the mycelial growth of S. sclerotiorum up to 80% and the sclerotial germination 0-100%. In the greenhouse pot test of ten isolates conducted in summer, 2 isolates B. megaterium (DK6) and B. cereus (C210) showed control efficacy on sclerotia viability of S. sclerotiorum, 20% and 35%, respectively. In the greenhouse pot test in winter, the disease incidence of the control group was 80%, whereas those of 9 isolates among 26 were approximately 20%. From the result, the 9 isolates are expected as potentially antagonistic bacteria for biological control of sclerotinia rot of lettuce caused by S. sclerotiorum.