• Fisheries and Aquatic Sciences
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• 제20권12호
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• pp.31.1-31.11
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• 2017

Background: Liver-expressed antimicrobial peptide-2 (LEAP-2) is an important component of innate immune system in teleosts. In order to understand isoform-specific involvement and regulation of LEAP-2 genes in mud loach (Misgurnus mizolepis, Cypriniformes), a commercially important food fish, this study was aimed to characterize gene structure and expression characteristics of two paralog LEAP-2 isoforms. Results: Mud loach LEAP-2 isoforms (LEAP-2A and LEAP-2B) showed conserved features in the core structure of mature peptides characterized by four Cys residues to form two disulfide bonds. The two paralog isoforms represented a tripartite genomic organization, known as a common structure of vertebrate LEAP-2 genes. Bioinformatic analysis predicted various transcription factor binding motifs in the 5'-flanking regions of mud loach LEAP-2 genes with regard to development and immune response. Mud loach LEAP-2A and LEAP-2B isoforms exhibited different tissue expression patterns and were developmentally regulated. Both isoforms are rapidly modulated toward upregulation during bacterial challenge in an isoform and/or tissue-dependent fashion. Conclusion: Both LEAP-2 isoforms play protective roles not only in embryonic and larval development but also in early immune response to bacterial invasion in mud loach. The regulation pattern of the two isoform genes under basal and stimulated conditions would be isoform-specific, suggestive of a certain degree of functional divergence between isoforms in innate immune system in this species.

• 한국어병학회지
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• 제25권3호
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• pp.159-164
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• 2012

Vibrio vulnificus ghosts (VVG) were generated using a mobilizable vector including a thermosensitive expression cassette by conjugation. The vaccine potential of VVG was investigated in mouse. Mice immunized with VVG showed significantly higher antibody titer than those with formalin-killed V. vulnificus. The present study supports the conceptive usefulness of bacterial ghosts as vaccine candidates.

• 자연과학논문집
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• 제4권
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• pp.1-10
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• 1991

발현 Vector인 pKK223-3를 이용하여 효모 Thiol-Specific Antioxidant단백질 유전자를 대장균에 도입시켜 이 단백질을 발현시켰다. 이 단백질은 대장균 단백질의 약 1% 정도로 발현되었으며, 물리 및 화학적 특성은 효모의 것과 동일한 특성을 보였다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.79.2-80
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• 2003

To better understand plant defense responses against pathogen attack, we identified the transcription factor-encoding genes in the hot pepper Capsicum annuum that show altered expression patterns during the hypersensitive response raised by challenge with bacterial pathogens. One of these genes, Ca1244, was characterized further. This gene encodes a plant-specific Type IIIA - zinc finger protein that contains two Cys$_2$His$_2$zinc fingers. Ca1244 expression is rapidly and specifically induced when pepper plants are challenged with bacterial pathogens to which they are resistant. In contrast, challenge with a pathogen to which the plants are susceptible only generates weak Ca1244 expression. Ca1244 expression is also strongly induced in pepper leaves by the exogenous application of ethephon, an ethylene releasing compound. Whereas, salicylic acid and methyl jasmonate had moderate effects. Pepper protoplasts expressing a Ca1244-smGFP fusion protein showed Ca1244 localizes in the nucleus. Transgenic tobacco plants overexpressing Ca1244 driven by the CaMV 355 promoter show increased resistance to challenge with a tobacco-specific bacterial pathogen. These plants also showed constitutive upregulation of the expression of multiple defense-related genes. These observations provide the first evidence that an Type IIIA - zinc finger protein, Ca1244, plays a crucial role in the activation of the pathogen defense response in plants.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.668-673
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• 2013

A recombinant E. coli co-expressing ALA synthase (hemA), NADP-dependent malic enzyme (maeB), and dicarboxylic acid transporter (dctA) was reported to synthesize porphyrin derivatives including iron-containing heme. To enhance the synthesis of bacterial heme, five genes of the porphyrin biosynthetic pathway [pantothenate kinase (coaA), ALA dehydratase (hemB), 1-hydroxymethylbilane synthase (hemC), uroporphyrinogen III synthase (hemD), and uroporphyrinogen III decarboxylase (hemE)] were amplified in the recombinant E. coli co-expressing hemA-maeB-dctA. Pantothenate kinase expression enabled the recombinant E. coli to accumulate intracellular CoA. Intracellular ALA was the most enhanced by uroporphyrinogen III synthase expression, porphobilinogen was the most enhanced by ALA dehydratase expression, uroporphyrin and coproporphyrin were the most enhanced by 1-hydroxymethylbilane synthase expression. The strain co-expressing coaA, hemA, maeB, and dctA produced heme of $0.49{\mu}mol/g$-DCW, which was twice as much from the strain without coaA expression. Further pathway gene amplifications for the porphyrin derivatives are discussed based on the results.

• Journal of Microbiology and Biotechnology
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• 제24권8호
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• pp.1051-1058
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• 2014

Platelet-activating factor receptor (PAFR) plays an important role in bacterial infection and inflammation. We examined the effect of the bacterial cell wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from Lactobacillus plantarum (pLTA) and Staphylococcus aureus (aLTA) on PAFR expression in THP-1, a monocyte-like cell line. LPS and aLTA, but not pLTA, significantly increased PAFR expression, whereas priming with pLTA inhibited LPS-mediated or aLTA-mediated PAFR expression. Expression of Toll-like receptor (TLR) 2 and 4, and CD14 increased with LPS and aLTA treatments, but was inhibited by pLTA pretreatment. Neutralizing antibodies against TLR2, TLR4, and CD14 showed that these receptors were important in LPS-mediated or aLTA-mediated PAFR expression. PAFR expression is mainly regulated by the nuclear factor kappa B signaling pathway. Blocking PAF binding to PAFR using a PAFR inhibitor indicated that LPS-mediated or aLTA-mediated PAF expression affected TNF-${\alpha}$ production. In the mouse small intestine, pLTA inhibited PAFR, TLR2, and TLR4 expression that was induced by heat-labile toxin. Our data suggested that pLTA has an anti-inflammatory effect by inhibiting the expression of PAFR that was induced by pathogenic ligands.

• Journal of Microbiology and Biotechnology
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• 제26권5호
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• pp.938-945
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• 2016

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious threats to rice production. In this study, screening of rice for resistance to BLB was carried out at two different times and locations; that is, in a greenhouse during winter and in an open field during summer. The pathogenicity of Xoo race K1 was tested on 32 Korean rice cultivars. Inoculation was conducted at the maximum tillering stage, and the lesion length was measured after 14 days of inoculation. Five cultivars, Hanareum, Namcheon, Samgdeok, Samgang, and Yangjo, were found to be resistant in both the greenhouse and open-field screenings. Expression of the plant defense-related genes JAmyb, OsNPR1, OsPR1a, OsWRKY45, and OsPR10b was observed in resistant and susceptible cultivars by qRT-PCR. Among the five genes tested, only OsPR10b showed coherent expression with the phenotypes. Screening of resistance to Xoo in rice was more accurate when conducted in open fields in the summer cultivation period than in greenhouses in winter. The expression of plant defense-related genes after bacterial inoculation could give another perspective in elucidating defense mechanisms by using both resistant and susceptible individuals.

• Archives of Pharmacal Research
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• 제28권4호
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• pp.433-437
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• 2005

Human cytochromes P450 (GYP) 2A6 and 2E1 are of great interest because of their important roles in the oxidation of numerous drugs and carcinogens. Bacterial expression systems, especially Escherichia coli cells, have been widely used for the production of various GYP enzymes in order to obtain high yield of proteins. The expression methods usually employ longer culture time (30-72 h) at lower temperature (usually under $30^{\circ}C$). Expression levels of GYPs 2A6 and 2E1 at $37^{\circ}C$ were compared to those at $28^{\circ}C$, which is a usual temperature used in most bacterial expression systems for human GYP expression. Within 18 h the expression levels of GYPs 2A6 and 2E1 reached up to 360 and 560 nmol per liter culture at $37^{\circ}C$, respectively, which are compatible with those of 36 h culture at $28^{\circ}C$. The activities of GYPs expressed at $37^{\circ}C$ were also comparable to those expressed at $28^{\circ}C$. The present over-expression system can be useful for rapid production of large amounts of active human GYPs 2A6 and 2E1 in E. coli.

• Molecules and Cells
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• 제19권2호
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• pp.232-238
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• 2005

Corneal endothelial cells play an important role in maintaining the transparency and ionic balance of the cornea. Inflammation causes many changes in the intracellular and extracellular environment of the cornea, including acidosis. We examined the relationship between changes in extracellular pH and expression of cyclooxygenase-2 in cultured bovine corneal endothelial cells. When extracellular pH ($[pH]_o$) was reduced to pH 6.4, COX-2 mRNA increased, with a peak at 2 h. This was blocked by pretreatment with actinomycin D and incubation with spermine NONOate (SPER/NO, a nitric oxide donor). Exposure to the $H^+$ ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), also raised COX-2 mRNA levels. CCCP-induced COX-2 mRNA expression was also reduced by SPER/NO. These results were confirmed immuno-cytochemically. These data demonstrate that COX-2 expression is stimulated by the lowering of extracellular pH that could result from bacterial infection, and that this is countered by over-production of nitric oxide, which could also result from bacterial infection.

• BMB Reports
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• 제30권1호
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• pp.60-65
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• 1997

Expression of human Cu.Zn-superoxide dismutase (SOD) with activity comparable to human erythrocyte enzyme was achieved in E. coli B21(DE3) by using the pET-17b expression vector containing a T7 promoter. Recombinant human SOD was found in the cytosol of disrupted bacterial cells and represented > 25% of the total bacterial proteins. The protein produced by the E. coli cells was purified using a combination of ammonium sulfate precipitation, Sephacryl S-100 gel filtration and DEAE-Sephacel ion exchange chromatography. The recombinant Cu,Zn-SOD and human erythrocyte enzyme were compared using dismutation activity, SDS-PAGE and immunoblotting analysis. The mass of the subunits was determined to be 15,809 by using a electrospray mass spectrometer. The copper specific chelator. diethyldithiocarbamate (DOC) reacted with the recombinant Cu,Zn-SOD. At $50{\mu}M$ and $100{\mu}M$ concentrations of DOC, the dismutation activity was not inhibited for one hour but gradually reduced after one hour. This result suggests that the reaction of DOC with the enzyme occurred in two distinct phases (phase I and phase II). During phase I of this reaction, one DOC reacted with the copper center, with retention of the dismutation activity while the second DOC displaced the copper, with a loss of activity in phase II.