• 한국가금학회지
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• 제44권2호
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• pp.93-102
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• 2017

본 연구에서 NDV의 L유전자와 F유전자를 표적 부위로 각각 제작한 primer 세트를 사용함으로써 하나의 PCR 튜브에서 NDV 검출(386 bp의 증폭 크기)과 함께 병원성 NDV(229 bp의 증폭 크기)를 동시에 감별 증폭할 수 있는 dRT-PCR 검사법을 개발하였다. 개발된 dRT-PCR검사법은 NDV를 특이적으로 검출하고, 병원성을 감별하였다. 특히 국내 병성감정 실시기관에서 적용 중인 기존의 RT-PCR 상용키트에서는 검출하지 못하는 class I NDV과 PPMV(class II 유전형 VI형)을 NDV를 검출함과 동시에 병원성 NDV도 감별가능하였다. 개발된 dRT-PCR 검사법의 검출 민감도는 약 $10^{3.0}EID_{50}/0.1mL$로 평가되었다. 또한 ND발생국의 야외 시료에 적용했을 때, NDV 공통항원 검출율은 94.4%였으며, 병원성 NDV 검출율은 100%이었다. 그러므로 본 연구에서 개발한 dRT-PCR 검사법은 의심축 사례에서 ND를 신속 정확하게 진단하는 데 유용할 진단 방법을 제공할 수 있을 것으로 판단된다.

• Tuberculosis and Respiratory Diseases
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• 제50권1호
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• pp.94-105
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• 2001

연구배경 : Pyrazinamide(PZA)는 결핵의 1차 치료 약제이며, 산성 환경 하에서만 작용하기 때문에 in vitro에서 생물학적인 감수성 검사를 하는 것이 어렵다. pncA는 PZase를 생성하는 유전자로 이의 돌연변이가 결핵균의 PZase 활성을 소실시켜 결핵균이 PZA에 대해서 내성을 획득하게 되는 기전으로 추정된다. 본 연구에서는 PZA의 생물학적 감수성, PZase 활성 및 pncA 유전자의 돌연변이 사이의 관계를 검토하고, 아울러 결핵균의 pncA 의 염기서열 결정이 PZA 내성을 예측하는데 이용될 수 있는지를 알아보고자 하였다. 방법 : 폐결핵 환자의 객담에서 분리된 결핵균 28균주를 대상으로 하여, 절대농도법으로 PZA의 감수성을 검사하고, pncA 유전자의 open reading frame 전체 561 bp를 포함하는 710 bp 크기의 유전자를 선택적으로 증폭하였다. PCR 증폭 산물을 직접 염기서열 결정에 이용하였고, GenBank에서 확인한 결핵균 야생주의 pncA 염기서열과 비교하여 돌연변이 여부를 확인하였다. 결과 : PZase 활성이 있는 6균주 모두 pncA의 돌연변이가 없었으나, 1균주(16.7%)는 절대농도법에 의한 감수성 검사에서 위내성으로 나타났다. PZase 활성이 없는 22균주 중 21균주(95.5%)는 pncA의 돌연변이가 확인되었고, 생물학적 감수성 검사에서는 20균주가 PZA 내성, 1균주는 검사 불능, 1균주는 감수성을 보였다. pncA의 돌연변이의 양상(중복 1예 포함)은 promotor 지역과 pncA의 전 open reading frame에 걸쳐서 단일 염기의 치환에 의한 silent mutation 1개, missense mutation 10개, nonsense mutation 1개, 3염기가 삽입된 insertion 1개, 1~3개의 염기가 삽입된 frame shift mutation 4개, 그리고 2~2347개의 염기가 결실된 frame shift mutation 2개였다. 그리고 나머지 3균주는 모두 pncA유전자의 open reading frame 상부의 11번째 염기가 똑같이 adenine에서 guanine으로 치환되어 있었다. 결론 : pncA의 돌연변이는 결핵균의 PZase 활성의 소실에 의한 PZA 내성의 주요 기전이며, 특히 pncA 시작 codon의 상부 11 번째 위치의 염기 치환은 promotor의 돌연변이에 의한 PZA 내성의 주요 부위인 것으로 추정된다. Automatic sequencing에 의한 pncA의 염기서열 결정은 결핵균의 PZA 내성 진단을 위한 빠르고 효과적인 방법으로 이용할 수 있으며, PZA 내성 결핵균의 균주간 연관성을 규명하기 위한 역학 조사의 목적으로 이용할 수 있을 것으로 생각된다.

• Journal of Applied Biological Chemistry
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• 제66권
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• pp.204-212
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• 2023

미백은 멜라닌 세포 내 멜라닌 생성 억제 기능을 의미한다. 기존 미백소재의 피부 부작용 때문에 최근에는 천연 소재를 활용한 미백 연구가 활발히 진행되고 있다. 무화과(Ficus Carica L.)는 뽕나무과에 속하는 열매로 줄기와 잎 성분의 미백 활성은 보고되었으나 무화과 열매의 미백 활성은 알려지지 않아 본 연구를 통해 멜라닌 생성 억제, 항산화 및 항염증 활성을 규명하고자 하였다. 무화과 열매 추출물(Figs fruits extract, FFE)의 라디칼 소거 활성은 DPPH/ABTS 분석에서 최대 농도에서 대조군 대비 34.52±1.98%/60.71±1.26% 수준으로 관찰되었다. CCK-8 assay를 통한 FFE의 세포독성은 약 10% 농도부터 관찰 되어 독성이 없는 최대 농도를 5%로 설정하여 모든 실험에 적용하였다. FFE는 inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6 및 tumor necrosis factor-α 유전자 발현 억제와 함께 NO 생성을 농도 의존적으로 감소시켜 항염증 활성이 있음을 알 수 있었다. 또한 미백 기능 규명을 위해 α-MSH로 자극된 B16F10 세포에서 FFE를 농도별로 처리한 결과 세포 내 멜라닌 생성을 유의하게 하향 조절했을 뿐만 아니라 시험관 내에서 tyrosinase 활성이 억제되었다. 또한 FFE는 RT-PCR에서 α-MSH 처리군에 비해 Microphthalmia-associated transcription factor (MITF) mRNA 발현을 약 94.34% 감소시켰다. 마지막으로, FFE는 α-MSH로 자극된 B16F10 세포에서 MITF, cAMP response element-binding protein 및 tyrosinase 단백질 발현을 유의하게 감소시켰다. 이러한 결과를 통해 우리는 FFE가 tyrosinase 효소 활성을 직접적으로 억제할 수 있을 뿐만 아니라 α-MSH 신호 기전 내 MITF 유전자 발현 조절을 통해 멜라닌 생성을 억제할 수 있음을 확인하였다.

• 한국어병학회지
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• 제20권2호
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• pp.119-127
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• 2007

Phosphoinositide-specific phospholipase Cδ (PLCδ) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to characterize the catalytic and regulatory properties of the PLCδ of Misgurnus mizolepis (ML-PLCδ). The ML-PLCδ gene was cloned and expressed under according to the method of the previous report (Kim et al., 2004), and its recombinant protein was purified by successive chromatography using Ni2+-NTA affinity column. The recombinant ML-PLCδ showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidylinositol (PI). Its activity was absolutely Ca2+-dependence, which was similar to mammalian PLCδ isozymes. The Ca2+ concentration yielding maximal activation of ML-PLCδ was 100 μM. However, the activity was decreased interestingly by a polyamine, such as spermine and spermidine. In vitro assay using cholate-micelle cell, ML-PLCδ activity was inhibited in dose-dependent manner by sphinogosine but increased by phosphocholine . In the lipid-binding assay, ML-PLCδ was strongly bound to LPA, PI(3)P, PI(4)P, PI(5)P, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3 and PA, but it showed the low affinity to S1P, PI(3,4)P2 and PS. Taken together our results, it is suggested that the general catalytic and regulatory properties of ML-PLCδ are similar with those of mammalian PLCδ1 isozymes, but the N-terminal extended piscine phospholipase Cδ1 (ML-PLCδ) might reflect some distinctions in regulatory properties and inositol-lipid binding specificity between piscine ML-PLCδ and mammalian PLCδ isozymes.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.61-61
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• 2001

The main goal of this study was to produce transgenic piglets by the method of injection of sperm-mediated exogenous DNA. Spermatozoa (1$\times$106 sperm of final concentration) obtained from caudal epididymis were mixed with pBC1-hEPO (20 ng/${mu}ell$) or pcDNA3 LAC Z (20 ng/${mu}ell$), and followed by electroporation (500 V, 25 ㎌). Matured oocytes having the first polar body and dense cytoplasm were selected and centrifuged at 12,000g for 6 min. After sperm injection, the oocytes were activated electrically (1.7 ㎸/cm, 30 $\mu$ sec, single pulse) in 0.3 M mannitol solution. Eggs injected sperm were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air for 192 h. This study were comprised 3 experiments. Experiment 1 compared the developmental efficiencies between the sperm-injected oocytes (Group 1) and further activated electrically (Group 2). Experiment 2 compared the expression of pcDNA3 LAC Z in the embryos produced by Group 1 and Group 2. Finally, experiment 3 carried out transfer of embryos (1-8 cell stage) transfected with pBC1 -hEPO into surrogate recipients synchronized by injection of combination of PG600 with hCG. The rates of cleavage and development into blastocyst stage in Group 2 were significantly higher than those of Group 1 (71.3% and 28.1% vs. 43.3% and 10.3%, respectively, p<0.05). Thirty (24.2%) out of 124 embryos analyzed in Group 2 were positive by X-gal. Similarly, in Group 1, 16.3% (8/49) were positive. After transfer of 789 embryos to 7 recipient gilts, three out of them examined by ultrasound became pregnant. One recipient is in day 50 pregnancy. On day 54 of gestation, two were carried out uterotomy in order to confirm the pregnancy One had 7 and another had 2 fetuses. We conclude that injection of sperm-mediated gene transfer will be used as a valuable tool for the production of transgenic piglets.

• Asian-Australasian Journal of Animal Sciences
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• 제25권2호
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• pp.194-199
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• 2012

Liver and pulmonary artery tissue from 5 Angus cross bred steers, 6 goats, and 6 pigs were collected at a commercial abattoir to examine the relationship of pulmonary artery copper (Cu) concentrations and genes involved in copper homeostasis. Liver and pulmonary artery samples were collected at the time of harvest and snap frozen. Liver and pulmonary artery Cu concentrations were determined via flame atomic absorption spectrophotometry and gene expression was determined via real time PCR. Liver Cu concentrations (mg Cu/kg DM${\pm}$SE) were higher (p<0.01) in cows ($396.4{\pm}109.1$) and goats ($181.4{\pm}37.0$) than in pigs ($19.2{\pm}3.5$). All liver Cu concentrations were within normal ranges and considered adequate for each species. Liver Cu concentration was more variable in cows and goats compared to pig liver Cu concentrations. Pulmonary artery ${\beta}$-hydroxylproline was higher (p<0.01) in cow and pig than goat. Real Time PCR revealed that goat liver atp7a was positively correlated ($r^2$ = 0.92; p<0.01) to liver Cu concentrations while cow and pig atp7a was not correlated to liver Cu concentration. In the pig, liver atp7a concentration was positively correlated to atp7b ($r^2$ = 0.66; p<0.05). Pulmonary artery Cu concentration was highest in cows ($14.9{\pm}4.7$), intermediate in pigs ($8.9{\pm}3.3$), and lowest in goats ($3.9{\pm}1.1$). Goat pulmonary artery Cu concentration was not correlated to ctr1 concentration, however, atp7a concentration was positively correlated with ctr1 ($r^2$ = 0.90; p<0.01). In cow pulmonary artery, loxl1 concentration was positively correlated to eln mRNA concentration ($r^2$ = 0.91; p<0.02). Pulmonary artery CTR1 protein concentration was positively correlated to pulmonary artery Cu ($r^2$ = 0.85; p = 0.03) concentration while negatively correlated to liver Cu ($r^2$ = -0.79; p<0.04). Pulmonary artery Cu concentration was not correlated to concentration of Cu homeostatic genes in the pig. These data indicate that genes involved in Cu homeostasis (ctr1, atp7A, atp7B, loxl1 and eln) are differently regulated in different species.

• Asian-Australasian Journal of Animal Sciences
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• 제29권11호
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• pp.1555-1561
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• 2016

Shank skin color of Korean native chicken (KNC) shows large color variations. It varies from white, yellow, green, bluish or grey to black, whilst in the majority of European breeds the shanks are typically yellow-colored. Three shank skin color-related traits (i.e., lightness [$L^*$], redness [$a^*$], and yellowness [$b^*$]) were measured by a spectrophotometer in 585 progeny from 68 nuclear families in the KNC resource population. We performed genome scan linkage analysis to identify loci that affect quantitatively measured shank skin color traits in KNC. All these birds were genotyped with 167 DNA markers located throughout the 26 autosomes. The SOLAR program was used to conduct multipoint variance-component quantitative trait locus (QTL) analyses. We detected a major QTL that affects $b^*$ value (logarithm of odds [LOD] = 47.5, $p=1.60{\times}10^{-49}$) on GGA24 (GGA for Gallus gallus). At the same location, we also detected a QTL that influences $a^*$ value (LOD = 14.2, $p=6.14{\times}10^{-16}$). Additionally, beta-carotene dioxygenase 2 (BCDO2), the obvious positional candidate gene under the linkage peaks on GGA24, was investigated by the two association tests: i.e., measured genotype association (MGA) and quantitative transmission disequilibrium test (QTDT). Significant associations were detected between BCDO2 g.9367 A>C and $a^*$ ($P_{MGA}=1.69{\times}10^{-28}$; $P_{QTDT}=2.40{\times}10^{-25}$). The strongest associations were between BCDO2 g.9367 A>C and $b^*$ ($P_{MGA}=3.56{\times}10^{-66}$; $P_{QTDT}=1.68{\times}10^{-65}$). However, linkage analyses conditional on the single nucleotide polymorphism indicated that other functional variants should exist. Taken together, we demonstrate for the first time the linkage and association between the BCDO2 locus on GGA24 and quantitatively measured shank skin color traits in KNC.

• 대한환경공학회지
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• 제29권5호
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• pp.556-563
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• 2007

고전압 방전극이 기체상에 위치하고 접지 전극이 수중에 설치된 기체-액체 혼합 방전에 의한 화학적 활성종의 발생 특성에 관해 실험실 규모 실험을 수행하였다. 실험된 전극 구조는 기존의 연구에서 사용해왔던 일반적 전극 배열에서보다 높은 전계 강도(electric field strength)를 형성하고 짧은 폭을 지닌 펄스들을 생성시킴으로써 방전에 의해서 일어나는 화학반응의 에너지 효율성을 높일 수 있는 것으로 나타났다. 방전에 의해 기체상에 생성되는 오존 농도는 실험된 전압 범위의 중간 값인 45 kV 조건에서 가장 높은 것으로 관찰되었다. 용액 전도도가 낮을수록 액체상을 통한 전기 저항이 증가하여 기체상에서 높은 전계 강도가 형성되므로 오존 생성을 촉진시키는 것으로 나타났다. 인가전압이 증가할수록 높은 전계 강도가 형성되어 강한 방전이 이루어지므로 과산화수소 생성속도가 증가하는 것으로 나타났다. 낮은 전압에서는 용액 전도도가 증가하면 과산화수소 분해속도가 증가하기 때문에 과산화수소 생성 속도가 감소하며 높은 전압에서는 용액 전도도가 증가하면 자외선 조사 등에 의해 과산화수소 발생의 중간 생성물인 OH 라디칼의 발생이 촉진되므로 과산화수소 생성 속도가 증가하는 것으로 나타났다. 산소와 아르곤의 혼합기체가 공급될 때, 강하고 안정한 방전이 이루어져 과산화수소 생성속도가 증가하는 것으로 나타났다.

• 대한임상검사과학회지
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• 제53권1호
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• pp.41-48
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• 2021

Essential thrombocythemia (ET) is a clonal bone marrow stem cell disorder, primarily involving the megakaryocytic lineage. The WHO 2016 guidelines include the molecular detection of JAK2, MPL, and CALR mutations as a major diagnostic criterion for ET. This study aimed to determine the frequency of JAK2 V617F, MPL W515L, and CALR mutations in Iraqi Kurdish patients afflicted with ET, and to analyze their clinical and hematological features. A total of 73 Iraqi Kurdish patients with ET were enrolled as subjects, and analysis was achieved utilizing real-time PCR. The frequency of JAK2 V617F, CALR, and MPL W515L mutations was determined to be 50.7%, 22%, and 16.4%, respectively. No statistically significant difference was obtained when considering the age and gender among different genotypes. The JAK2 V617F mutated patients had significantly higher white blood cell counts and hemoglobin levels than the CALR-positive patients (P-value=0.000, 0.007, respectively), MPL W515L-positive patients (P-value=0.000, 0.000, respectively), and triple negative patients (P-value=0.000, 0.000, respectively). Also, the JAK2 V617F mutated patients showed higher platelet count as compared to the MPL W515L-positive patients (P-value=0.02) and triple negative patients (P-value=0.04). Furthermore, significantly lower white blood cell count and hemoglobin levels were associated with CALR positivity (P-value=0.000, 0.01, respectively), MPL W515L-positivity (P-value=0.001, 0.000, respectively), and triple negativity (P-value=0.000, 0.000, respectively), as compared to patients with combined mutations. In conclusion, apart from a relatively high frequency of MPL W515L mutation, our data is comparable to earlier reports, and highlights the importance of genotyping the JAK2 V617F, MPL W515L, and CALR mutations for accurate diagnosis of patients with ET.

• Molecules and Cells
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• 제45권7호
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• pp.465-478
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• 2022

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of target messenger RNA (mRNA) complementary to the 3' untranslated region (UTR) at the post-transcriptional level. Hsa-miR-422a, which is commonly known as miRNA derived from transposable element (MDTE), was derived from short interspersed nuclear element (SINE). Through expression analysis, hsa-miR-422a was found to be highly expressed in both the small intestine and liver of crab-eating monkey. AT-Rich Interaction Domain 5 B (ARID5B) was selected as the target gene of hsa-miR-422a, which has two binding sites in both the exon and 3'UTR of ARID5B. To identify the interaction between hsa-miR-422a and ARID5B, a dual luciferase assay was conducted in HepG2 cell line. The luciferase activity of cells treated with the hsa-miR-422a mimic was upregulated and inversely downregulated when both the hsa-miR-422a mimic and inhibitor were administered. Nuclear factor erythroid-2 (NF-E2) was selected as the core transcription factor (TF) via feed forward loop analysis. The luciferase expression was downregulated when both the hsa-miR-422a mimic and siRNA of NF-E2 were treated, compared to the treatment of the hsa-miR-422a mimic alone. The present study suggests that hsa-miR-422a derived from SINE could bind to the exon region as well as the 3'UTR of ARID5B. Additionally, hsa-miR-422a was found to share binding sites in ARID5B with several TFs, including NF-E2. The hsa-miR-422a might thus interact with TF to regulate the expression of ARID5B, as demonstrated experimentally. Altogether, hsa-miR-422a acts as a super enhancer miRNA of ARID5B by collaborating with TF and NF-E2.