• The Plant Pathology Journal
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• 제31권2호
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• pp.108-114
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• 2015

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about $6.39{\times}10^5$ transformants/$3{\mu}g$ pGADT7-Rec. The titer of the primary cDNA library was $2.5{\times}10^8cfu/mL$. The numbers for the cDNA library was $2.46{\times}10^5$. Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

• The Korean journal of internal medicine
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• 제34권1호
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• pp.146-155
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• 2019

Background/Aims: Indoxyl sulfate (IS) is a uremic toxin and an important causative factor in the progression of chronic kidney disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to exhibit protective effects in kidney injury. Here, we investigated the effects of paricalcitol treatment on IS-induced renal tubular injury. Methods: The fluorescent dye 2',7'-dichlorofluorescein diacetate was used to measure intracellular reactive oxygen species (ROS) following IS administration in human renal proximal tubular epithelial (HK-2) cells. The effects of IS on cell viability were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays and levels of apoptosis-related proteins (Bcl-2-associated protein X [Bax] and B-cell lymphoma 2 [Bcl-2]), nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) p65, and phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase B (Akt) were determined by semiquantitative immunoblotting. The promoter activity of $NF-{\kappa}B$ was measured by luciferase assays and apoptosis was determined by f low cytometry of cells stained with f luorescein isothiocyanate-conjugated Annexin V protein. Results: IS treatment increased ROS production, decreased cell viability and induced apoptosis in HK-2 cells. IS treatment increased the expression of apoptosis-related protein Bax, decreased Bcl-2 expression, and activated phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt. In contrast, paricalcitol treatment decreased Bax expression, increased Bcl-2 expression, and inhibited phosphorylation of MAPK, $NF-{\kappa}B$ p65, and Akt in HK-2 cells. $NF-{\kappa}B$ promoter activity was increased following IS, administration and was counteracted by pretreatment with paricalcitol. Additionally, flow cytometry analysis revealed that IS-induced apoptosis was attenuated by paricalcitol treatment, which resulted in decreased numbers of fluorescein isothiocyanate-conjugated Annexin V positive cells. Conclusions: Treatment with paricalcitol inhibited IS-induced apoptosis by regulating MAPK, $NF-{\kappa}B$, and Akt signaling pathway in HK-2 cells.

• 대한환경공학회지
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• 제32권3호
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• pp.248-255
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• 2010

본 연구는 연안해역에서 발생하는 적조의 현장 제어기술로 전자빔을 이용하고자 적조사멸에 대한 전자빔 조사의 효율성을 평가하였다. Prorocentrum minimum, Prorocentrum micans, Cochlodinium polykrikoides, Heterosigma akashiwo, Alexnadrium catenella의 5종을 실험대상 적조로 선정하여 배양하였으며, 실제 경남 통영(2007. 8. 15)에서 발생한 적조에 대해서도 동일한 실험을 수행하였다. 전자빔 조사선량은 1 kGy, 2 kGy, 4 kGy, 8 kGy로 1 kGy의 전자빔 조사로 50~65%의 적조사멸율을 보였으며, 1일 경과 시 86~97% 사멸하였다. Chlorophyll-a의 경우 조사 즉시 50~64% 감소하고, 1일 경과한 뒤 72~93%의 감소율을 보였다. 또한 전자빔 조사 즉시 S-protein은 28~47%, S-carbohydrate는 16~45% 가 증가하였으며, 1일의 시간 경과 후에는 S-protein이 77~138%, S-carbohydrate가 77~212%까지 증가하였다. 또한 투과 전자현미경을 통해 전자빔 조사 전후의 적조세포 관측을 통하여 세포의 파괴 및 세포내 구성물질의 용출을 확인할 수 있었다. 이렇듯 적조에 전자빔을 조사함으로써 Extracellular Biopolymer에 의한 Flocculation을 유도하여 적조를 침강 제어할 수 있다. 또한 전자빔 조사 전 후 Alexandrium catenella의 PSP(Paralytic Shellfish Poisoning) 패독 분석 결과 1 kGy의 전자빔을 조사함으로써 전자빔을 조사하지 않은 적조 대비 PSP 독성 함량이 48% 감소하여 전자빔조사가 유독적조의 사멸 뿐만 아니라 제독에도 효과가 있음을 알 수 있었다.

• 한국환경보건학회지
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• 제29권1호
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• pp.74-83
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• 2003

Microcystin, stable compounds with circular heptapeptides, is presented inside cyanobacterial cell. So far, over 30 types have been known to exist and microcystin-LR, RR among them are the most potent toxin compound. By this reason, a strong oxidant, ozone was used in this study to remove the microcystins produced by cyanobacteria. Removal efficiency of microcystin at M water treatment plant was also evaluated. Microcystin concentration was determined by protein phosphatase inhibition assay. The results showed that dissolved microcystin in raw water detected in the range of 0.011-0.028 ㎍ Microcystin-RR equivalent/l. Above 98% of microcystin was removed through overall treatment system. Therefore, the water treatability of M treatment plant seemed to be excellent. Removal efficiency of microcystin according to unit process varied as characteristics of raw water such as DOC, UV/sub 254/ and turbidity. Removal efficiency of microcystin by ozonation was investigated in laboratory according to contact time and ozone dose. Dissolved microcystin was increased by twice fold according to ozone contact time, but increased by fifth fold according to ozone dose. So, changing of ozone dose more affected microcystin release than changing of ozone contact time. Behavior of microcystin by ozonation was similar to that of DOC, and residual ozone concentration gave influence to removal ratio of microcystin. In conclusion, single ozone treatment wasn't effective on microcystin removal in case of water containing a lot of cells. Therefore, it's more effective to use ozonation process after the removal of cyanobacterial cells in advance.

• KSBB Journal
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• 제6권3호
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• pp.289-297
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• 1991

The insecticidal protein gene in the pKL-20-1 clone derived from Bacillus thuringiensis serovar. kurstaki plasmid was subcloned in the plant shuttle vector, pGA643. The 7.3 kb fragment was cloned in the BglII and Hpal sites of pGA643 vector and expressed in E. coli S17-1, which produced insecticidal proteins killing Bombyx mori larvae. The clone was named pHL-20. The protoplast formation, calli induction and plantlet regeneration of Nicotiana tabacum was carried out. A tremendous number of mesophyll protoplasts of N. tabacum were formed, up to 7$\times$105 protoplast per ml, for 20 hours in darkness in the enzyme solution of 0.5% cellulase and 0.1% macerosin, pH 5.8. The viabilities of the protoplasts were maintained above 80% for 6 days in the media containing 2mg/1 of NAA and 1mg/1 of kinetin. Calli were induced from the protoplasts and leaves of the N. tabacum on MS medium containing 0.5mg/1 BAP. Under the culture conditions the protoplasts underwent repeated cell division into calli. Plantlets were regenerated from callus cultures derived from protoplast and leaves. Shoots were induced in a medium containing 1mg/1 of BAP.

• 한국미생물·생명공학회지
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• 제18권6호
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• pp.647-652
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• 1990

Bacillus thuringiensis subsp. kurstaki HD1의 내 독소생산과 내독소단백질 유전자의 클로닝에 관한 연구를 하였다. 상기 균주는 아포생성기간 중에 이중피라미드형의 내독소를 생산하였고, 크리는 약 $2.9\times 1.0 \mu m$이었다. 상기 균주는 약 10개의 플라수미드 DNA를 가지고 있었으며, 플라스미드의 분자량의 범위는 2.1에서 80kilobases였다. 플라스미드 73kb, BamHI 절단 29Kb DNA 단편과 PstI 절단 7.9Kb DNA는 Probe DNA와 혼성화되었다. PstI 7.9Kb DNA를 추출하여 운반체인 pBR322 운반체의 PstI 절단부위에 삽입하여 클로닝한 후에 E.coli HB101 균주에 형질전환하였으며, 이 클로운을 pKL-20-1로 명명했고 이 형질전환체는 Bombyxmori 유충을 치사시키는 독소물질을 생산하였다.

• IMMUNE NETWORK
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• 제12권6호
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• pp.261-268
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• 2012

Respiratory syncytial virus (RSV) and influenza virus are the most significant pathogens causing respiratory tract diseases. Composite vaccines are useful in reducing the number of vaccination and confer protection against multiple infectious agents. In this study, we generated fusion of RSV G protein core fragment (amino acid residues 131 to 230) and influenza HA1 globular head domain (amino acid residues 62 to 284) as a dual vaccine candidate. This fusion protein, Gcf-HA1, was bacterially expressed, purified by metal resin affinity chromatography, and refolded in PBS. BALB/c mice were intranasally immunized with Gcf-HA1 in combination with a mucosal adjuvant, cholera toxin (CT). Both serum IgG and mucosal IgA responses specific to Gcf and HA1 were significantly increased in Gcf-HA1/CT-vaccinated mice. To determine the protective efficacy of Gcf-HA1/CT vaccine, immunized mice were challenged with RSV (A2 strain) or influenza virus (A/PR/8/34). Neither detectable viral replication nor pathology was observed in the lungs of the immune mice. These results demonstrate that immunity induced by intranasal Gcf-HA1/CT immunization confers complete protection against both RSV and homologous influenza virus infection, suggesting our Gcf-HA1 vaccine candidate could be further developed as a dual subunit vaccine against RSV and influenza virus.

• 한국미생물·생명공학회지
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• 제50권3호
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• pp.387-394
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• 2022

Verotoxin-1 (VT-1) or Shiga-like toxin 1 (Stx-1) is produced by enterohemorrhagic Escherichia coli (EHEC) and is an AB5 holotoxin with a strong inhibitor of protein synthesis. VT-1 is a type 2 ribosome-inactivating protein (RIP) that has been shown to have cytotoxic and anticancer potential by inducing necrosis, apoptosis, and cell cycle arrest, making it a promising antitumor candidate. Here, we tested the cytotoxicity of VT-1 on CaCo2 and NCM425 cell lines and the results showed that VT-1 was more potent on CaCo2. Morphological changes were also evaluated on the cellular level and the results showed that VT-1 caused a decrease in viable cell count, altered cell membrane permeability, and an increase in total nuclear intensity. On the other hand, VT-1 displayed a lesser impact on mitochondrial membrane potential (MMP) and cytochrome c release. On the expression of caspases 3 and 9, VT-1 exhibited an insignificant effect on both which alongside the mitochondrial membrane potential (MMP) and cytochrome c results, might indicate that CaCo2 suffered from the necrosis process as a mechanism of cell death after exposure to VT-1.

• The Korean Journal of Physiology and Pharmacology
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• 제19권6호
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• pp.549-555
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• 2015

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors /PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

• 대한의생명과학회지
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• 제12권3호
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• pp.145-151
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• 2006

Leukotactin-l (Lkn-l )/CCL15 has been known as a potent chemoattractant of leukocytes. However, the precise function of Lkn-l in human neutrophils has not been explained well. In the present study, we investigated the contribution of Lkn-1 in chemotactic activity of human neutrophils. Both CCR1 and CCR3 mRNA expressions are strongly expressed in human neutrophils but CCR2 protein expression was uniquely detected on the cell surface. Lkn-l binding to CCR1 and CCR3 induced chemotactic activity of neutrophils. Chemotactic index of Lkn-l was comparable to that of IL-8. $MIP-1{\alpha}/CCL3$ binding to CCR1 and CCR5 has no effect on neutrophil migration. Cell migration, in response to Lkn-l, was blocked by pertussis toxin (Ptx), a $G_o/G_i$ protein inhibitor, and U73122, a phospholipase C(PLC) inhibitor but not by protein kinase C inhibitor such as rottlerin, and Ro-31-8425. Taken together, our results demonstrate that Lkn-l transduces the chemotaxis signal through $G_o/G_i$ protein and PLC. This finding provides the molecular mechanism by which Lkn-l may contribute to neutrophil movement into the site of inflammation.