• 대한의생명과학회지
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• 제29권1호
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• pp.48-52
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• 2023

Formaldehyde use is associated with serious health risks, which can affect medical personnel and technicians. Therefore, we investigated the efficacy of an alternative fixative, with respect to two types of formalin fixatives, by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, immunohistochemical (IHC) staining, and RNA extraction. For H&E staining, the circular nucleus was stained dark blue by the basic dye hematoxylin and the cytoplasm was stained red by the acid dye eosin in all three fixative samples. No difference was found in the Duksan General Science (DGS), Sigma-Aldrich, and Core-Fix fixative samples (Corebiotech) used to fix kidney tissue, after PAS staining. IHC staining showed that CD4 was significantly increased in the lippolysaccharide (LPS)-treated group compared to the control group (vehicle), confirming the changes in specific molecules. The quantity and quality of RNA from tissues fixed in the three types of fixatives were evaluated. The average concentration of RNA was 106 ng/µL and average purity at A 260/280 ratio was 1.7~2.0, regardless of fixative used. For quality of protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was confirmed by Western blotting. In conclusion, Core-Fix can be used as a fixative for pathological tissues, in histological and molecular diagnoses.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 추계학술대회
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• pp.143-143
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• 2022

Plant defense systems against pathogens have been studied extensively and are currently a hot topic in plant science. Using a reverse genetics technique, this study looked into the involvement of the NO-downregulated NAD(P)-binding Rossmann-fold superfamily gene in plant growth and defense in Arabidopsis thaliana. For this purpose, the knockout and overexpressing plant of the candidate gene along with the relevant controls were exposed to control, oxidative and nitro-oxidative stresses. The results showed that candidate gene negatively regulates plants' root and shoot lengths. To investigate the role of the candidate gene in plant basal defense, R-gene-mediated resistance and systemic acquired resistance (SAR) plants were challenged with virulent or avirulent strains of Pseudomonas syringae pathovar tomato (Psf) DC3000. The results showed that the candidate gene negatively regulates plants' basal defense, R-gene-mediated resistance and SAR. Further characterization via GO analysis associated the candidate gene with metabolic and cellular processes and response to light stimulus, nucleotide binding and cellular location in the cytosol and nucleus. Protein structure analysis indicated the presence of a canonical Oxidoreductase family NAD (P)-binding Rossmann fold domain of 120 amino acids with a total of 121 plant homologs across 35 different plant species in the clad streptophyta. Arabidopsis eFP browser showed its expression in almost all the above-ground parts. Protein analysis indicated C225 and C359 as potential targets for S-Nitrosylation by NO. SMART analysis indicated possible interactions with mevalonate/galactokinase, galacturonic acid kinase, arabinose kinase, putative xylulose kinase, GroES-like zinc-binding alcohol dehydrogenase and various glyceraldehyde-3-phosphate dehydrogenases.

• Journal of Veterinary Science
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• 제24권5호
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• pp.72.1-72.16
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• 2023

Background: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface of Streptococcus dysgalactiae, coded with gapC, is a glycolytic enzyme that was reported to be a moonlighting protein and virulence factor. Objective: This study assessed GAPDH as a potential immunization candidate protein to prevent streptococcus infections. Methods: Mice were vaccinated subcutaneously with recombinant GAPDH and challenged with S. dysgalactiae in vivo. They were then evaluated using histological methods. rGAPDH of mouse bone marrow-derived dendritic cells (BMDCs) was evaluated using immunoblotting, reverse transcription quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay methods. Results: Vaccination with rGAPDH improved the survival rates and decreased the bacterial burdens in the mammary glands compared to the control group. The mechanism by which rGAPDH vaccination protects against S. dysgalactiae was investigated. In vitro experiments showed that rGAPDH boosted the generation of interleukin-10 and tumor necrosis factor-α. Treatment of BMDCs with TAK-242, a toll-like receptor 4 inhibitor, or C29, a toll-like receptor 2 inhibitor, reduced cytokines substantially, suggesting that rGAPDH may be a potential ligand for both TLR2 and TLR4. Subsequent investigations showed that rGAPDH may activate the phosphorylation of MAPKs and nuclear factor-κB. Conclusions: GAPDH is a promising immunization candidate protein for targeting virulence and enhancing immune-mediated protection. Further investigations are warranted to understand the mechanisms underlying the activation of BMDCs by rGAPDH in a TLR2- and TLR4-dependent manner and the regulation of inflammatory cytokines contributing to mastitis pathogenesis.

• IMMUNE NETWORK
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• 제23권4호
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• pp.31.1-31.18
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• 2023

Evidence suggests that the human respiratory tract, as with the gastrointestinal tract, has evolved to its current state in association with commensal microbes. However, little is known about how the airway microbiome affects the development of airway immune system. Here, we uncover a previously unidentified mode of interaction between host airway immunity and a unique strain (AIT01) of Staphylococcus epidermidis, a predominant species of the nasal microbiome. Intranasal administration of AIT01 increased the population of neutrophils and monocytes in mouse lungs. The recruitment of these immune cells resulted in the protection of the murine host against infection by Pseudomonas aeruginosa, a pathogenic bacterium. Interestingly, an AIT01-secreted protein identified as GAPDH, a well-known bacterial moonlighting protein, mediated this protective effect. Intranasal delivery of the purified GAPDH conferred significant resistance against other Gram-negative pathogens (Klebsiella pneumoniae and Acinetobacter baumannii) and influenza A virus. Our findings demonstrate the potential of a native nasal microbe and its secretory protein to enhance innate immune defense against airway infections. These results offer a promising preventive measure, particularly relevant in the context of global pandemics.

• 한국균학회지
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• 제52권1호
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• pp.55-60
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• 2024

2023년 8월 청주 땅콩 재배포장에서 기보고되어 있는 점무늬 병징과는 다른 양상의 잎 반점을 발견하였다. 갈색 반점은 주로 잎 가장자리에 형성되었으며, 반점 부위에서 원인균을 분리하였다. 순수 분리한 병원균을 땅콩 유묘에 인공접종하여 병원성 검정을 수행하였다. 그 결과, 접종 5-7일 뒤에 유사한 병징을 확인할 수 있었고 병징 부위에서 동일한 균을 재분리하였다. 분리한 균주의ACT-CHS-1-GAPDH-ITS-TUB2 영역을 이용하여 동정 및 유연관계 분석을 수행한 결과 C. sojae임을 확인하였다. 따라서 본 연구는 국내 최초로C. sojae에 의해 발생한 땅콩 탄저병을 보고하고자 한다.

• 한국작물학회지
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• 제67권4호
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• pp.211-221
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• 2022

논 토양 조건에서 팥의 생육기 중 초기 생육 단계에 해당되는 유묘기에 과습 스트레스 처리 후 생육특성 변화 및 팥잎의 단백질 발현 양상을 조사한 결과는 다음과 같다. 1. 아라리(밀양 8호)를 공시 품종으로 과습 스트레스 실험을 진행하였다. 3일간 과습 처리하는 동안 팥의 초장과 생체중(잎, 줄기)은 통계적으로 유의하지는 않았으나 생체중(뿌리)와 SPAD value 값은 통계적으로 유의한 결과가 나왔다. 2. 과습 처리 3일 후 잎의 단백질 발현 양상을 확인한 결과 21개의 차별 발현된 단백질 중 과습 스트레스 처리에 의해 9개의 단백질들이 up-regulated 되었고, 12개의 단백질들이 down-regulated 되었다. 단백질을 기능별로 분류한 결과 Biological Process에서 carbohydrate metabolic process와 관련된 단백질들이 가장 많이 나왔고 Cellular Component에서는 Chloroplast에서 가장 많이 존재하였다. 3. Regulatory protein과 관련된 Maturase K 단백질은 처리구에서 대조구보다 발현양이 증가한 것으로 나타났다. 4. Carbohydrate metabolic process와 관련된 Malate dehydrogenase 단백질과 Glyceraldehyde-3-phosphate dehydrogenase 단백질은 과습 스트레스를 받았을 경우 단백질 발현양이 증가하였다. 5. Photosynthesis와 관련된 Carbonic anhydrase (CA)는 처리구보다 대조구에서 단백질 발현량이 더 증가한 것을 확인할 수 있었다. 스트레스와 관련된 단백질 Superoxide dismutase는 처리구에서 대조구보다 단백질 발현양이 증가한 것을 확인할 수 있었다.

• 대한한방내과학회지
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• 제32권1호
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• pp.33-42
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• 2011

Background : Despite recent advances in cancer management, prognosis of ovarian cancer is poor. Anticancer effects of herbal medicine, such as Euonymus alatus Sieb, Oldenlandia diffusa (Willd.) Roxburgh, and Orostachys japonicus A. Berger, have been reported in treatment of ovarian and cervical cancers, but the systematic approaches to explain their molecular mechanism(s) have not yet been established. Objectives : To establish a basis of understanding for anti-cancer mechanisms of herbal medicine, we profiled protein expression in human ovarian and cervical cancer cells treated with the extracts from Euonymus alatus Sieb, Oldenlandia diffusa (Willd.) Roxburgh and Orostachys japonicus A. Berger. Methods : Human ovarian cancer cell line NIH:OVCAR-3, and human cervical cancer cell line HeLa were employed in the present study. Whole protein was obtained from the cells harvested at 48 hours after the treatment with herbal water-extract, and analyzed by 2DE-based proteomic approach. Results : Various changes of protein expression induced by the herbal treatment were monitored : down-regulation of molecular chaperone (calreticulin variant), glycolytic enzymes (D-3-phosphoglycerate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase and alpha-enolase), RNA processing molecules (hnRNP A2/B1), and antioxidant protein (peroxiredoxin 1). Conclusions : Repression of glycolysis has been accepted as the mechanism to increase anticancer reagent's effect. Thus, down-regulation of glycolytic enzymes by the herbal extracts suggested a possible synergistic effect of herbs in the presence of platinum-based therapeutics. In further study, as well as the synergistic effect of the herbs, it has to be further validated whether artificial regulation of hnRNP A2/B1 in ovarian cancer cells affects various cancer survival factors, since RNA processing can be interrupted by deranged expression of hnRNP subtypes, and it results in an inhibition of cancer cell growth.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.915-921
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• 2001

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.

• Asian-Australasian Journal of Animal Sciences
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• 제28권11호
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• pp.1649-1656
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• 2015

Gene expression profiling has offered new insights into postmortem molecular changes associated with meat quality. To acquire reliable transcript quantification, high quality RNA is required. The objective of this study was to analyze integrity of RNA isolated from chicken skeletal muscle (pectoralis major) and its capability of serving as the template in quantitative real-time polymerase chain reaction (qPCR) as a function of postmortem intervals representing the end-points of evisceration, carcass chilling and aging stages in chicken abattoirs. Chicken breast muscle was dissected from the carcasses (n = 6) immediately after evisceration, and one-third of each sample was instantly snap-frozen and labeled as 20 min postmortem. The remaining muscle was stored on ice until the next rounds of sample collection (1.5 h and 6 h postmortem). The delayed postmortem duration did not significantly affect $A_{260}/A_{280}$ and $A_{260}/A_{230}$ ($p{\geq}0.05$), suggesting no altered purity of total RNA. Apart from a slight decrease in the 28s:18s ribosomal RNA ratio in 1.5 h samples (p<0.05), the value was not statistically different between 20 min and 6 h samples ($p{\geq}0.05$), indicating intact total RNA up to 6 h. Abundance of reference genes encoding beta-actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine-guanine phosphoribosyltransferase (HPRT), peptidylprolylisomerase A (PPIA) and TATA box-binding protein (TBP) as well as meat-quality associated genes (insulin-like growth factor 1 (IGF1), pyruvate dehydrogenase kinase isozyme 4 (PDK4), and peroxisome proliferator-activated receptor delta (PPARD) were investigated using qPCR. Transcript abundances of ACTB, GAPDH, HPRT, and PPIA were significantly different among all postmortem time points (p<0.05). Transcript levels of PDK4 and PPARD were significantly reduced in the 6 h samples (p<0.05). The findings suggest an adverse effect of a prolonged postmortem duration on reliability of transcript quantification in chicken skeletal muscle. For the best RNA quality, chicken skeletal muscle should be immediately collected after evisceration or within 20 min postmortem, and rapidly preserved by deep freezing.

• KSBB Journal
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• 제17권2호
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• pp.162-168
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• 2002

본 연구에서는 유도성 promoter인 GAL1과 상시성 promoter인 GPD와 ADH1 promoter 하류에 사람 ferritin H-chain 유전자(hfL) 및 사람 ferritin L-chain 유전자(hfL)를 연결하여 재조합 plasmid를 구축하고 이들을 S. cerevisiae 2805에 형질 전환시켜 외래 유전자를 성공적으로 발현시켰다. Ferritin 발현에 미치는 promoter의 영향을 비교한 바, 이 두 단백질 생간에 있어서 GAL1 promoter가 GPD나 ABH1 promoter 보다 더 효율적이었다. GAL1 promoter를 사용한 형질 전환체 (YG-H와 YG-L)에서 H-chain의 발현율은 전체 수용성 단백질 중 4.5%에 해당하였고, L-chain의 발현율은 9.8%에 이르렀다. 각 균주에서 발현된 H 및 L subunit은 비변성 젤은 사용한 전기 영동의 결과 대장균에서 생산된 재조합 단백질과 마찬가지로 자발적으로 holoprotein으로 조합되어졌다. 재조합 H-와 L-ferritin들은 단백질 내공에 철을 축적할 수 있음이 Prussian blue 염색으로 확인되었다. 그리고 효모 세포를 10 mM ferric citrate를 함유한 배지에서 배양했을 때, H-ferritin과 L-ferritin을 생산하는 재조합 효모 균주에 있어서의 펄의 농도는 각각 174.9 $\mu\textrm{g}$ Per gram(dry cell weight)과 148.8 $\mu\textrm{g}$ Per gram(dry cell weight)이었고 야생형 효모 균주에 있어서의 털의 농도는 49.4 $\mu\textrm{g}$ Per gram(dry cell weight)이었다. 이것은 사람 ferritin 유전자를 효모 균주에 발현시킴으로써 효모의 철 함량이 증진되었음을 유추하는 결과이다.