Anti-inflammatory effects of osthole in peripheral blood mononuclear cells from Hanwoo (Bos taurus coreanae)

김승창,이승환,채한화,김의형,정기용,장선식,최봉환 충남대학교 농업과학연구소 2019 Korean Journal of Agricultural Science Vol.46 No.3

Due to the ban on the use of antibiotics, interest has been increasing for the development of therapeutic agents to treat various diseases using natural resources. Osthole, a natural coumarin compound used in traditional Chinese medicines, exerts an anti-inflammatory effect, but its effects in cows remain unknown. In this study, the effect of osthole on lipopolysaccharide (LPS)- or concanavalin-A (Con-A)- stimulated peripheral blood mononuclear cells (PBMCs) was assessed. Jugular venous blood was collected from Korean calves, and PBMCs were isolated. They were then used to study the immune response of PBMCs to treatment with osthole and LPS or Con-A for 72 h by measuring inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Osthole significantly inhibited the mRNA secretion of TNF-α and IFN-γ in a dose-dependent manner. Therefore, osthole inhibited LPS- or Con-A- induced TNF-α and Con-A-induced IFN-γ production significantly in dose-dependent manner. These results clearly suggest that osthole inhibited the LPS- or Con-A- stimulated upregulation of pro-inflammatory cytokines in a dose-dependent manner, without causing obvious cytotoxic effects. Osthole could also protect cows from LPS- or Con-A- induced endotoxin shock, possibly by inhibiting the production of pro-inflammatory cytokines, which suggests that osthole might be a novel therapeutic agent for the prevention of inflammatory diseases.

단일염기다형성 마커를 이용한 백우 품종 식별 방법

김승창,김관우,노희종,김동교,김성우,김찬란,이상훈,고응규,조창연 한국산학기술학회 2020 한국산학기술학회논문지 Vol.21 No.1

This study was conducted to develop specific Single Nucleotide Polymorphism (SNP) markers to identify the genetic characteristics and breed of White Hanwoo (WH) using a molecular biological method. SNP genotyping was performed with an Illumina Bovine HD 777K SNP chip using DNA extracted from 48 Hanwoo and 22 WH. The minor allele frequency (MAF) difference of each SNP was calculated and the statistical significance (P-value) of the MAF difference was calculated through Fisher's Exact test (Genotype). SNPs with 100% difference in the MAF difference were selected based on marker selection criteria. The nine SNP markers with genetic differences were selected. The selected markers have different alleles as being Hanwoo- and WH- specific. Therefore, based on these results, it can be concluded that the Hanwoo and WH varieties can be clearly distinguished by using these SNPs. So, the patent of the WH breed identification markers was registered. WH is a breed that shows the characteristics of a Korean native species that is separate from the native Hanwoo. It is expected that genetic characteristics research on the WH can be used to identify the breed and as a knowledge base for enhancing the value of breeding stock. 본 연구는 백우 품종 육성을 위해 분자생물학적 방법을 이용하여 유전적 특성을 파악하고 백우 품종을 식별하기 위한 백우 품종 특이적인 Single Nucleotide Polymorphism (SNP) 마커를 개발하기 위해 수행되었다. 한우 48두와 백우 22두의 혈액에서 추출된 DNA를 이용하여 Illumina Bovine HD 777K SNP chip으로 SNP genotyping을 실시하였다. 각 SNP의 Minor Allele Frequency (MAF) difference (한우와 백우의 차이 절대값)을 계산하고, Fisher’s Exact test (Genotype)을 통해 MAF difference의 통계적 유의성(P-value)을 계산하였다. 품종 별 차이를 나타낼 수 있는 마커를 선발기준으로 MAF difference가 100%의 차이를 나타내는 SNP를 식별하였다. 이러한 유전적 차이를 보이는 9개의 단일염기다형성 마커(rs42125585, rs42125591, rs42125833, rs109461720, rs134735704, rs109447299, rs42164846, rs42160000 및 rs137353829)가 선발되었다. 선발된 마커들은 한우와 백우 특이적인 대립유전자를 가지고 서로 다른 대립유전자를 나타내고 있다. 이들 9개의 SNP 마커들을 이용하여 한우와 백우의 품종을 식별할 수 있음을 확인하였고, 이러한 결과들을 바탕으로 백우 품종 식별 마커 특허를 등록하였다. 백우는 원종인 한우에서 분리되어 한국 재래종의 특성을 잘 나타내 주는 계통으로, 이러한 백우가 가지고 있는 유전적 특성 연구는 백우를 식별하고 품종으로서 육종하는데 사용되어 종축으로서의 가치 증진을 위한 기반 연구가 될 것으로 생각된다.

Genomic Polymorphism Analysis using Microsatellite Markers in Gyeongju Donggyeong Dogs

김승창,김이경,최승규,박창민,박선애,조용민,임다정,최한하,이승환,이지웅,선상수,최봉환 사단법인 한국동물생명공학회 2013 Reproductive & developmental biology Vol.36 No.4

This study was conducted to find a useful marker for gene polymorphism analysis using Microsatellite marker (MS marker) in Gyeongju Donggyeong dog. Twenty three MS marker analyzed the genetic features of DNA using 100Gyeongju Donggyeong dogs in Gyeongju area. It was performed multiplex PCR with 3 set primer divided 9, 10 and 4 by analysis of conditions among MS markers. The results were calculated heterozygosity, polymorphic information content (PIC), allele frequency and number of allele at each locus using Microsatellite Toolkit software and Cervus 3.0 program. Total 148 alleles were genotyped to determine and average 6.43 alleles was detected. FH3381 had the highest of 15 alleles and FH2834 had the lowest of 2 alleles. Expected heterozygosity had a wide range from 0.282 to 0.876 and had average value of 0.6496. Also, Observed heterozygosity had a more wide range from 0.200 to 0.950 and had average value of 0.6404. PIC had range from 0.262 to 0.859 and average PIC was calculated 0.606. Especially, FH2998 represented the highest rate of observed heterozygosity of 0.950 and FH3381 represented the highest rate of expected heterozygosity of 0.876 and PIC of 0.859. The use of these markers was considered to be useful to study genetic traits of Gyeongju Donggyeong dog.

성염색체상의 MS 마커를 이용한 한국재래소의 유전적 특징

김승창 ( Seung-chang Kim ),조창연 ( Chang-yeon Cho ),최성복 ( Seong-bok Choi ),이지웅 ( Ji-woong Lee ),김재환 ( Jae-hwan Kim ) 한국국제농업개발학회 2015 韓國國際農業開發學會誌 Vol.27 No.5

이 연구의 목적은 성염색체 특이적 초위성체마커의 대립 유전자 다양성과 빈도를 조사하여 한국 재래소 3품종의 (칡소,한우 그리고 제주흑우) 유전적 근연관계 및 특징을 조사하여 우리 고유 유전자원으로서의 가치를 구명하고자 하였다. 한국재래소 3품종은 30개의 초위성체 마커에서 선별된 4개의 초위성체 마커(INRA30, TGLA325, UMN0905 및 UMN0929)를 이용하여 식별하였다. 대립 유전자의 다양성, 대립 유전자빈도, 이형접합도 그리고 다형 정보량(PIC)을 산출 하였다. 칡소, 제주 흑우와 한우에 대한 관찰 이형접합도의 평균은 각각0.541, 0.406 및 0.61이었고 PIC의 평균은 각각 0.542, 0.377그리고 0.6이었다. 제주흑우의 경우 칡소나 한우에 비해 낮은 이형접합도와 PIC를 보여준다. 이들 4개의 초위성체 마커를 이용하여 칡소, 제주흑우 그리고 한우의 품종을 구별에 이용할 수 있다. 이러한 결과들로 볼 때 한국 재래소 3품종은 가축유전자원으로서 중요한 가치를 지니고 있으며 이들 품종의 보존, 관리 및 활용에 중요한 기초자료로 이용될 것으로 사료된다. This study aimed to identify allele variability and determine the frequencies of microsatellite(MS) markers specific to bovine sex chromosomes in Korean native cattle (KNC; Chikso, Hanwoo and Jeju Black). The three KNC breeds were characterized using 4 MS markers (INRA30, TGLA325, UMN0905 and UMN0929), and allelic richness, allele frequency, heterozygosity and polymorphism information content (PIC) per locus were calculated. The mean observed heterozygosity and PIC values for Chikso, Jeju Black and Hanwoo were 0.541 and 0.542, 0.406 and 0.377, and 0.61 and 0.6, respectively. The heterozygosity and the PIC values of Jeju Black breed were smaller than those of Chikso and Hanwoo. Thus, these genetic parameters were used for the characterization of the breeds and for determining their genetic relationships. The findings of this study might be useful for the conservation, management, and utilization of KNC breeds as genetic resources.

단일염기다형성 마커를 이용한 칡소 품종의 식별

김승창(Seungchang Kim),김관우(Kwan-Woo Kim),이은도(Eun-Do Lee),진대혁(Daehyeok Jin),김동교(Dongkyo Kim),이진욱(Jinwook Lee),최봉환(Bong-Hwan Choi),김재환(Jae-Hwan Kim) 한국산학기술학회 2022 한국산학기술학회논문지 Vol.23 No.2

한우의 말초혈액 단핵세포 염증성 사이토카인에 대한 브로피리민의 효과 확인

김승창 ( Seungchang Kim ),이승환 ( Seung-Hwan Lee ),채한화 ( Han-Ha Chai ),김의형 ( Ui-Hyung Kim ),정기용 ( Ki-Yong Chung ),임다정 ( Dajeong Lim ),박종은 ( Jong-Eun Park ),조용민 ( Yongmin Cho ),최봉환 ( Bong-Hwan Choi ) 한국축산학회(구 한국동물자원과학회) 2017 동물자원연구 Vol.28 No.4

Bropirimine, a class of antineoplastic agents, is known as one of the potent immunomodulators and is currently under clinical development for the treatment of cancer. However, the effect of bropirimine on the cow remains unknown as a therapeutics agent. In this experiment, the effect of bropirimine in the peripheral blood mononuclear cells (PBMCs) stimulated by lipopolysaccharide (LPS) or concanavalin-A (Con-A) was examined. Jugular venous blood was collected from Korean Hanwoo calves and PBMCs were isolated. It was used to study the effect of bropirimine upon stimulation with LPS or Con-A for 72 hours. The expression pro-inflammatory cytokines like Tumor Necrosis Factor α (TNF-α) and Interferon γ (IFN-γ) were confirmed. Bropirimine significantly inhibited LPS- or Con-A-induced TNF-α and Con-A-induced IFN-γ in dose-dependent manner. Furthermore, Bropirimine inhibited TNF-α and Con-A mRNA expression at the transcription level. These results clearly indicated that bropirimine inhibited LPS or Con-A stimulated up-regulation of proinflammatory cytokines in a dose-dependent manner without conspicuous cytotoxicity. The bropirimine has potential to protect cow from LPS or Con-A induced endotoxin shock, possibly through inhibition of the production of proinflammatory cytokines. It suggesting that bropirimine may be a novel therapeutic agent for the prevention of inflammatory diseases. This result revealed specific features of the immune responses depending on the bropirimine compound and would help to knowledge of bovine immunity.

Genetic Diversity Analysis of Kyrgyz Native Horse and Korean Horses using Microsatellite Markers

Seungchang Kim(김승창),Bolot Toktosunov,Myrzakul Israilov,Daehyeok Jin(진대혁),HuiMang Song(송희망),Jae-Yeong Lee(이재영),Sangrae Cho(조상래),Bong-Hwan Choi(최봉환),Sungsil Moon(문성실),Jae-Young Choi(최재영),Asankadyr Zhunu 한국산학기술학회 2023 한국산학기술학회논문지 Vol.24 No.10

키르기즈 재래마는 키르기즈 공화국에서 경제적으로나 전통적으로 중요한 가축 중 하나이며 키르기즈스탄 사람들의 역사와 가치를 대표하고 있다. 이 연구의 목적은 국내 말 품종과의 비교 분석을 통해 키르기스 재래마의 유전적 다양성, 구조 및 특성을 파악하기 위한 것이다. 19개의 초위성체 마커를 사용하여 키르기스 재래마와 한국 품종 3종(제주마, 제주 교배종, 서러브레드)의 특성을 분석했다. 관찰된 평균 이형접합도와 기대 이형접합도는 각각 0.689, 0.794로 나타났다. 키르기즈 재래마의 평균 대립유전자 수는 9.474개였다. 다형성정보량(PIC)은 6개의 유전자좌(AHT4, TKY297, VHL20, LEX3, ASB17, HTG10)에서 높은 다형성(> 0.8)을 나타냈다. 계통 발생학적 분석, 요인 대응 분석(FCA), 혼합도 분석 결과 네 품종이 네 가지 집단으로 분리된 것으로 나타났다. 이러한 결과를 바탕으로 키르기즈 재래 마의 지속적인 보존을 위해 근친교배를 고려한 꾸준한 관리 계획 교배가 필요할 것으로 고려되며, 향후 유전자원 유지 및 보전 계획 수립의 기초자료로 활용될 수 있을 것으로 생각된다. The Kyrgyz native horse (KH) is one of the economically and traditionally important livestock in the Kyrgyz Republic, representing the history and values of the Kyrgyz people. The Objectives of this study were to determine the genetic diversity, structure, and characteristics of KH through comparative analysis with Korean horse breeds. The KH and three Korean breeds (Jeju horse, Jeju crossbred, throughbred) were characterized by using 19 microsatellite markers. The mean observed and expected heterozygosities were 0.689 and 0.794, respectively. The mean number of alleles for the KH was 9.474. The polymorphism information content (PIC) showed that six loci (AHT4, TKY297, VHL20, LEX3, ASB17 and HTG10) were highly polymorphic (> 0.8). The Phylogenetic analysis, factorial correspondence analysis (FCA) and admixture analysis showed that four breeds were separated into four different populations. Based on these results, it is considered that steady management planned breeding considering inbreeding is necessary for the continued presevation of KH, and it could be used as a basis for future genetic resource maintenance and conservation planning.

Development of Microsatellite Markers for Discriminating Native Korean and Imported Cattle Breeds

Seungchang Kim(김승창),Chang-Yeon Cho(조창연),Hee-Jong Roh(노희종),Seong-Heum Yeon(연성흠),Seong-Bok Choi(최성복) 한국생명과학회 2017 생명과학회지 Vol.27 No.4

성염색체에 위치하는 5 개의 초위성체 마커(INRA30, TGLA325, UMN0803, UMN0905, UMN0929) 를 이용하여 재래소 3품종과 외래소 7품종(칡소, 한우, 제주흑우, 홀스타인, 일본화우, 샤롤레, 앵거스, 헤어포드, 시멘탈, 한우X샤롤레 교잡종)의 유전적 특징을 확인하였다. 상업적으로 판매되는 소고기의 잘못된 원산지 표기를 통해 부당한 경제적 이득을 취하고자 하는 문제를 해결하기 위한 방법으로 소고기 샘플을 빠르고 저비용으로 확인 하기 위한 방법으로 사용하기 위해 좌위 또는 품종 특이적 대립유전자를 탐색하고 좌위별 대립유전자수, 대립유전자빈도, 이형접합도 그리고 다형정보량(PIC)을 구하여 이들 10품종의 유전적 다양성을 평가하였다. STRUCTURE 분석을 통한 군락의 분류 및 유전적 균일성 분석에서 재래소 품종과 외래소 품종으로 두개의 주요 그룹으로 나뉘어진다. 이러한 결과들은 재래소와 외래소 품종의 특이적인 유전적 차이를 나타낸다. 또한 Nei’s 표준 유전적 거리로 나타난 neighbor-joining tree에서도 독립적인 계통유전학적인 위치를 보여주었다. 이러한 결과는 국내 재래종과 외래품종 사이의 유전적 거리, 품종의 역사 및 그들의 지리적 기원 사이에 명백한 차이를 나타내는 증거로 사료된다. 이러한 결과들로 이들 성염색체의 초위성체 마커들에 의해 소 품종들의 유전적 다양성과 연관성은 과학적인 기초자료로 활용되고 재래소와 외래품종 소고기를 구별할 수 있는 DNA 마커들로 이용될 수 있을 것으로 사료된다. 그러므로 이러한 마커들은 효율적인 이력추적 시스템을 만드는데 사용되어 원산지 표시 위반을 억제하는데 유용할 것이다. Three Korean native cattle (KNC) and seven exotic breeds (Chikso, Hanwoo, Jeju black, Holstein, Japanese black, Charolais, Angus, Hereford, Simmental, and Cross breed) were characterized by using five microsatellite (MS) markers (INRA30, TGLA325, UMN0803, UMN0905, and UMN0929) from the sex chromosome. Genetic diversity was evaluated across the 10 breeds by using the number of alleles per locus, allele frequency, heterozygosity, and polymorphism information content (PIC) to search for locus and/or breed specific alleles, allowing a rapid and cost-effective identification of cattle samples, avoiding mislabeling of commercial beef. It was divided into two main groups from STRUCTURE analysis, one corresponding to KNC and the other to exotic cattle breeds. These results also showed specific genetic differences between KNC and exotic breeds. Nei’s standard genetic distance was calculated and used in the construction of a neighbor-joining tree. Results evidenced a correspondence between genetic distance, breeds’ history, and their geographic origin, and a clear separation between KNC and exotic breeds. Overall, this study evidenced that DNA markers can discriminate between domestic and imported beef, contributing to the knowledge on cattle breeds’ genetic diversity and relationships by using MS markers of the sex chromosome. These markers would be useful for inhibitory effect about false sales and for building an effective tracking system.

Effect of Endocrine Disruptors on the Oocyte Maturation and Ovulation in Amphibians, Rana dybowskii

최미정,김승창,김안나,권혁방,안련섭 한국통합생물학회 2007 Animal cells and systems Vol.11 No.1

Recently, we have shown that some endocrinedisruptors, heavy metals, organotins and azoles suppressedsteroidogenic enzymes such as P450 side-chain cleavageenzyme (P450sc) and aromatase in bullfrog ovarian follicles.In the present study, by using an amphibian ovarian follicleculture system, we examined the effects of these endocrinedisruptors on maturation and ovulation of oocytes fromRana dybowskii in vitro. Ovarian fragments or isolatedfolicles were cultured for 24 h in a medium containing frogpituitary homogenate (FPH) or progesterone (P4) with orwithout endocrine disruptors, and oocyte maturation (germinalvesicle breakdown, GVBD) and ovulation were examined.Among the organotins, tributyltin (TBT) strongly inhibitedboth FPH- and P4-induced oocyte maturation (ED500.7M, respectively); however, tetrabutyltin (TTBT) anddibutyltin (DBT) showed only partial suppression, whilemonobutyltin (MBT) showed no inhibitory effect. All of theorganotins suppressed P4-induced oocyte ovulation veryeffectively at a low concentration, and TBT and DBT exertedan inhibitory effect on FPH-induced ovulation. Among theheavy metals, mercury (Hg), cadmium (Cd) and cobalt (Co)were very effective in inhibiting FPH-induced oocytematuration and ovulation, while lead (Pb), arsenite (As) andmetals suppressed FPH-induced oocyte ovulation at a highdose (100 M). Among the azoles, itraconazole (ICZ),ketoconazole (KCZ) and clotrimazole (CTZ) effectivelyinhibited FPH-induced oocyte maturation and ovulation,while econazole (ECZ), miconazole (MCZ) and fluconazole(FCZ) were considerably less effective. These resultsdemonstrated that the abovementioned endocrine disruptorsexhibited diferential effects on oocyte maturation andovulation in amphibian follicles and that the frog ovarianculture system could be used as an effective experimentaldisruptors in vitro.

한우, 칡소 및 제주 흑우 Calpain-Calpastatin 유전자 다양성

이승환,김승창,조수현,최봉환,Aditi Sharma,임다정,당창권,장선식,김재환,고문석,양보석,강희설 충남대학교 농업과학연구소 2013 농업과학연구 Vol.40 No.2

The aim of study was to investigate genetic diversity for the calpain/calpastatin gene in three Hanwoo breeds [(Brown (n=62), Brindle (n=81) and Jeju Black (n=30)]. Random samples from three breeds of Hanwoo were selected and genotyped for the 7 SNPs of calpain/calpastatin using TaqMan method. Allele frequencies were investigated for CAPN1/CAST gene. Allele frequency of CAST2 SNP was 0.75, 0.59 and 0.22 for Brown, Brindle and Jeju black, respectively. The CAST3 revealed allele frequency of 0.59 and 0.57 in Brown and Jeju Black, while it showed very low allele frequency (0.07) in Brindle. In particular, favorable allele (G allele) for the CAPN1-2 SNP which was shown a strong association with tenderness in Taurine and Indicine cattle revealed 16% and 17% higher allele frequency in Brown Hanwoo (0.82) comparing Brindle (0.66) and Jeju Black Hanwoo (0.65). AMOVA demonstrated that among population variance occupied only 10% of total variance and among individual variance was 0%, while within individual variance was 90% of total variance. This result showed that population effect contributed very small portion of genetic to these three Hanwoo breeds, while within individual variance contributed large portion of genetic diversity within these Hanwoo breeds. In conclusion, three Hanwoo breeds (Brown, Brindle and Jeju black) showed a genetically homogeneous based on the 7 SNPs of CAPN1/CAST gene and it came from same ancestor to form modern Hanwoo breed.