• 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.

• Clinical and Experimental Reproductive Medicine
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• 제28권3호
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• pp.183-190
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• 2001

Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

• 한국동물위생학회지
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• 제45권1호
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• pp.1-11
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• 2022

A novel porcine circovirus 4 (PCV4) was recently identified in Chinese and Korean pig herds. Although several conventional polymerase chain reaction (cPCR) and real-time PCR (qPCR) assays were used for PCV4 detection, more sensitive and reliable qPCR assay is needed that can simultaneously detect PCV4 and internal positive control (IPC) to avoid false-negative results. In the present study, a duplex qPCR (dqPCR) assay was developed using primers/probe sets targeting the PCV4 Cap gene and pig (glyceraldehyde-3-phosphate dehydrogenase) GAPDH gene as an IPC. The developed dqPCR assay was specifically detected PCV4 but not other PCVs and porcine pathogens, indicating that the newly designed primers/probe set is specific to the PCV4 Cap gene. Furthermore, GAPDH was stably amplified by the dqPCR in all tested viral and clinical samples containing pig cellular materials, indicating the high reliability of the dqPCR assay. The limit of detection of the assay 5 copies of the target PCV4 genes, but the sensitivity of the assay was higher than that of the previously described assays. The assay demonstrated high repeatability and reproducibility, with coefficients of intra-assay and inter-assay variation of less than 1.0%. Clinical evaluation using 102 diseased pig samples from 18 pig farms showed that PCV4 circulated in the Korean pig population. The detection rate of PCV4 obtained using the newly developed dqPCR was 26.5% (27/102), which was higher than that obtained using the previously described cPCR and TaqMan probe-based qPCR and similar to that obtained using the previously described SYBR Green-based qPCR. The dqPCR assay with IPC is highly specific, sensitive, and reliable for detecting PCV4 from clinical samples, and it will be useful for etiological diagnosis, epidemiological study, and control of the PCV4 infections.

• Genomics & Informatics
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• 제9권3호
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• pp.127-133
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• 2011

The Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) is one of the major genomic resources for human genetics and immunological studies. Use of LCLs is currently extended to pharmacogenetic studies to investigate variations in human gene expression as well as drug responses between individuals. We evaluated four common internal controls for gene expression analysis of selected hematopoietic transcriptional regulatory genes between B cells and LCLs. In this study, the expression pattern analyses showed that TBP (TATA box-binding protein) is a suitable internal control for normalization, whereas GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is not a good internal control for gene expression analyses of hematopoiesis-related genes between B cells and LCLs at different subculture passages. Using the TBP normalizer, we found significant gene expression changes in selected hematopoietic transcriptional regulatory genes (downregulation of RUNX1, RUNX3, CBFB, TLE1, and NOTCH2 ; upregulation of MSC and PLAGL2) between B cells and LCLs at different passage numbers. These results suggest that these hematopoietic transcriptional regulatory genes are potential cellular targets of EBV infection, contributing to EBV-mediated B-cell transformation and LCL immortalization.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.103-109
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• 1999

Objective: Heat shock protein 70-2 (Hsp70-2) gene knockout mice are found to have premeiotic arrest at the primary spermatocyte stage with a complete absence of spermatids and spermatozoa. This observation led to the hypothesis that hspA2 may be disrupted in human testes with abnormal spermatogenesis. To test this hypothesis, we studied the mRNA expression of hspA2 in infertile men with azoospermia. Design: The mRNA expression were analyzed by competitive RT-PCR among testes with normal spermatogenesis, pachytene spermatocyte arrest, and sertoli-cell only syndrome. Materials and methods: Testicular biopsy was performed in men with azoospermia (n=15). Specimens were subdivided into three groups: (group 1) normal spermatogenesis (n=5), (group 2) spermatocyte arrest (n=5), (group 3) Sertoli-cell only syndrome (n=5). Total RNA was extracted by Trizol reagent. Total extracted RNA was reverse transcribed into cDNA and amplified by PCR using specific primers for hspA2 target cDNAs. A competitive cDNA fragment was constructed by deleting a defined fragment from the target cDNA sequence, and then coamplified with the target cDNA for competitive PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Results: On Competitive RT-PCR analyses for hspA2 mRNA, significant amount of hspA2 expression was observed in group 1, whereas a constitutively low level of hspA2 was expressed in groups 2 and 3. Conclusion(s): The study demonstrates that the hspA2 gene expression is down-regulated in human testes with abnormal spermatogenesis, which in turn suggests that hspA2 gene may play a specific role during meiosis in human testes.

• 대한약리학회지
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• 제32권3호
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• pp.381-388
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• 1996

패혈증 환자에 흡입마취제를 투여하는 것은 혈역동학에 심각한 영향을 초래할 수 있다. 흡입마취제 투여시 내독소혈증흰쥐 대동맥 수축반응에 미치는 hydroxocobalamin의 영향을 알아보기위해, 내독소 (1.5 mg/kg)를 복강내 투여한 뒤 18시간 후에 대동맥을 적출하여 phenylephrine에 대한 수축반응과 이에 대한 흡입마취제 및 hydroxocobalamin의 작용을 알아보았다. 패혈증이 적절히 유발되었는 지를 확인하기 위해 RT-PCR을 이용하여 혈관 평활근에서 iNOS유도를 확인하였다. 내독소 처치에 의해 수축기 및 확장기 혈압이 대조군에 비해 유의하게 감소되었으나, 심박동수는 영향이 없었다. Phenylephrine에 대한 수축반응은 $10^{-8}\;M$부터 $10^{-5}\;M$까지 용량 의존적으로 증가했으며, 내독소혈증흰쥐 대동맥에서 수축반응이 억제되었다. halothane과 enflurane은 1 MAC 농도에서 phenylephrine에 의한 수축반응을 유의하게 억제했으나 isoflurane은 영향이 없었다. Hydroxocobalamin $(10^{-5}\;M)$은 흡입마취제의 종류 및 투여 유무에 관계없이 내독소혈증흰쥐 대동맥 수축반응을 증가시켰다. 이상의 결과로 미루어보아 hydroxocobalamin은 패혈증 환자의 흡입마취시 혈역동을 개선시킬 것으로 사료된다.

• 한국어류학회지
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• 제23권1호
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• pp.1-9
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• 2011

ACP (annealing control primer)를 사용하여 DDRT (differential display reverse transcription)-PCR 방법으로 볼락의 성장단계에 따라 발현량 차이를 나타내는 DEG (differentially expressed gene)를 확보하였다. ACP 120개를 분석하여 18개월령 근육조직에서보다 6개월령 근육조직에서 발현량이 많은 DEG 16개와 6개월령 근육조직에서보다 18개월령 근육조직에서 발현량이 더 많은 DEG22개의 염기서열을 분석하였다. DEG 염기서열을 BLAST 검색한 결과, parvalbumin (PVALB) 등 18개의 유전자(PVALB, NDKB, TPM, TnI, GAPDH, CKM2, factor 2 SERF2, AMPD, TRICA, ARHGAP15, ESD, hsp70, COL1A2, GST, Midllip1, MYL1, SERCA1B, FTH1)와 69~95%의 상동성을 나타냈다. Real time PCR 분석법으로 6개월령 근육조직에서 발현량이 많은 DEG14와 PVALB 유전자의 성장단계별 발현양상을 조사한 결과, 볼락이 성장함에 따라 발현량이 감소하였으며, 특히 PVALB 유전자는 6개월령 이후에는 발현량이 극히 적었다. 6개월령 근육조직에서보다 18 개월령 근육조직에서 발현량에서 많았던 CKM2 유전자는 성장함에 따라 발현량이 계속 증가하였고, 4세 이후에는 발현량이 감소하였다. DEG의 조직특이적 발현양상을 분석한 결과, DEG14는 근육, 간, 신장, 및 비장조직에서 발현되었으며, PVALB 유전자는 근육과 신장조직에서 발현되었고, 간과 비장조직에서는 발현되지 않았다. CKM2 유전자는 근육, 신장 및 비장조직에서 발현되었고, 간 조직에서는 발현되지 않았다. PVALB 유전자의 mRNA 크기는 659 bp 이며, 110개의 아미노산으로 구성되어 있다. Parvalbumin과 CKM2 유전자는 성장속도가 빠른 어류 선발에 이용할 수 있는 분자마커 개발에 활용하고자한다.

• 한국미생물·생명공학회지
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• 제48권1호
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• pp.79-89
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• 2020

본 연구는 단풍잎돼지풀 70% 에탄올 추출물의 항염증 활성을 검증하기 위해 수행되었다. 단풍잎돼지풀 70% 에탄올 추출물의 전자공여능 측정과 ABTS⁺ 라디칼 소거능을 측정한 결과, 1,000 ㎍/ml 농도에서 각각 84.1%와 92.5%의 효과를 나타냈고, 수렴활성 측정을 한 결과 1,000 ㎍/ml 농도에서 94.7%의 효과를 보였다. 단풍잎돼지풀 70% 에탄올 추출물의 항염증 효과를 측정하기 위해 lipopolysaccharide (LPS)로 유도된 RAW 264.7세포를 사용하여 효과를 측정하였다. 세포에서 단풍잎돼지풀 추출물의 세포독성을 측정하기 위해 MTT assay를 수행하였다. 그 결과, 500 ㎍/ml 농도에서 90% 이상의 생존율을 보였다. Nitric oxide 생산을 억제하는 효과를 측정한 결과, 단풍잎돼지풀 추출물에서 농도가 증가할수록 NO 생성이 감소되는 효과를 확인하였다. 단풍잎돼지풀 추출물의 단백질 발현효과를 western blot을 통해 25, 50, 100 ㎍/ml 농도에서 측정하였고, 양성 대조군으로 β-actin을 사용하였다. 그 결과, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 단백질 발현효과는 100 ㎍/ml 농도에서 8.6%, 25.1%의 감소됨을 확인하였다. ERK1/2, p38, JNK와 Iκ-Bα의 단백질발현 효과는 인산화를 통해 확인하였고, 농도의존적으로 감소하였음을 확인하였다. mRNA 발현 억제 효과를 RT-PCR을 통해 25, 50, 100 ㎍/ml의 농도에서 측정하였고, 양성 대조군으로 GAPDH를 사용하였다. 그 결과, LPS로 유도된 대식세포에서 iNOS, COX-2, interleukin (IL)-1β, IL-6, TNF-α의 mRNA 발현 억제 효과는 농도가 증가할수록 발현이 감소됨을 확인하였다. 결론적으로 단풍잎돼지풀 추출물은 염증을 억제할 수 있는 가능성이 있는 항염증 소재의 가능성을 증명하였다.