A Study of genetic polymorphisms of HLA-class I and II Genes Using Polymerase Chain Reaction

Lee,Kyung-Ok THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1998 Journal of biomedical laboratory sciences Vol.4 No.1

HLA 유전자군은 인간의 유전자 중에서 가장 높은 유전적 다형성을 보이며, 분석방법에 따라 판정할 수 있는 대립유전자의 수에 많은 차이가 있다. 현재까지 혈청학적 방법을 이용하여 HLA 항원형 구분을 하였으나, 최근 골수이식 등 여러 HLA 활용분야에서 HLA 유전자형 분석이 요구되고 있어, 많은 수의 HLA 유전자형을 쉽고 정확하게 구분할 수 있은 HLA DNA typing 방법이 필요한 실정이다. 본 연구에서는 HLA-A, B, C, DRB1 유전자형 구분은 PCR SSP 방법을, HLA-DQA1, DQB1, DPB1 유전자형 구분은 PCR-RFLP 방법을 사용한 HLA DNA typing 방법으로 한국인에서 HLA유전자형을 구분하고자 하였다. 본 방법을 이용하여 HLA형이 규명된 B-임파아구 표준세포에서 DNA typing을 실시하였을 때, 11차 국제조직적합성학회에서 발표된 결과와 모두 일치하였다. 한국인에서 HLA-A, -B, -C,대립유전자는 17종, 23종, 16종이 확인되었으며, HLA-DQA1, -DQB1, -DPB1, DRB1대립유전자는 8종, 16종, 13종, 37종이 확인되었다. 한국인에서 빈도가 높은 HLA-class Ⅰ유전자는 HLA-A 유전자에서 A*02가 27.0%였으며 HLA-B 유전자에서는 B*40이 17.6%를 나타내었고 HLA-C 유전자에서는 Cw*0101이 19.2%로 가장 높은 빈도를 나타내었다. 한국인에서 가장 빈도가 높은 HLA-classⅡ 유전자는 DQA1 유전자에서 DQA1*0301이 32.1%였고, DQB1 유전자에서는 DQB1*0303이 12.9%를 나타내었으며, DPB1 유전자에서는 DPB1*0501이 31.3%였고 DRB1 유전자에서는 DRB1*1501이 9.2%를 나타내었다. 본 연구에서 실시한 HLA DNA typing 방법은 비교적 빠른 시간내에 많은 종류의 HLA 대립유전자형을 정확하게 구분할 수 있으므로 앞으로 tissue typing 실험실에서 유용하게 활용될 수 있을 것으로 생각된다. 또한 DNA typing 방법을 이용하여 분석한 한국인의 HLA-classⅠ,Ⅱ 유전자군의 유전자형빈도는 골수이식을 비롯한 각종 이식검사, 특수 질환 관련검사나 인류유전학연구 등 HLA 유전자의 임상적 활용을 위한 자료로 사용될 수 있을 것으로 기대된다. The HLA genes located in the short arm of chromosome 6 specify heterodimeric glycoproteins involved in the regulation of the immune response. Recently, in the elucidation of HLA polymorphism, serological and cellular typing methods have been replaced by DNA typing using polymerase chain reaction (PCR). The purpose of this study was to establish the HLA DNA typing methods and determine gene frequencies of HLA molecules in Koreans. PCR-SSP(sequence specific primers) and PCR-RFLP (restriction fragment length polymorphism) techniques were used for the analysis of HLA-A, -B, -C, DRB1 genes and HLA-DQA1, DQB1, DPB1 genes, respectively. The results of B-lymphoblastoid cells used for control experiment were consistent with the previous data identified in the 11th International Histocompatibility Workshop. Seventeen, 23, 16, 8, 16, 13 and 37 types of HLA-A, B, C, DQA1, DQB1, DPB1 and DRB1 alleles were found, respectively, in a total of unrelated 120 Korean individuals. The most frequent HLA alleles were A*02 (27.0%), B*40 (17.6%), Cw*01 (19.2%), DQA1*0301 (32.1%), DQB1*0303 (12.9%), DPB1*0501 (31.3%) and DRB1*1501 (9.2%) among Koreans. Theis study shows that DNA typing method using PCR technique is a relatively simple, fast and practical tool for the determination of the HLA-class Ⅰand Ⅱ genes. Moreover, the data of HLA gene frequencies could be useful for the Korean database before clinical applications, including organ and unrelated bone marrow transplantation, anthropological study, disease association and individual identification.

DNA중합효소 연쇄반응을 이용한 한국형 젖소 면역결핍 바이러스의 검출

권오식 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1999 Journal of biomedical laboratory sciences Vol.5 No.1

젖소 면역결핍 바이러스 (BIV)는 젖소에게 여러 가지 면역결핍증후군을 야기하는 렌티바이러스 (역전사효소 바이러스의 아군)로서 국내에서는 이에 대한 연구나 보고가 진행된 바 없다. 본 연구에서는 BIV가 다른 젖소의 역전사효소 바이러스인 젖소 백혈병바이러스 (BLV)와 동시 감염되고 (5% 정도)있는 점을 착안해 우선 대구·경북지역의 소의 BLV 감염실태를 조사해 BLV 가 감염된 젖소를 대상으로 BIV 감염상태를 알아보았다. 먼저 10회에 걸쳐 젖소와 황소 248마리의 혈액을 채취해 BLV와 BIV의 숙주세포가 되는 말초혈액 단핵구 (peripheral blood monocytes, PBMC)를 분리하고, 이를 이용해 DNA 중합효소 연쇄반응 (PCR)과 Southern blot 분석법을 통해 BLV와 BIV의 존재여부를 조사하였다. 그 결과 조사대상 젖소의 66.9%(81/121) 이상이 BLV에 감염되어 있음을 PCR 검사를 통해 알 수 있었으며, 이는 Southern blot법으로 재차 확인되었다. 이 결과는 종래에 보고된 대구·경북지역에서의 BLV 감염율인 27∼30%보다 2배 이상 높은 수치로서, (1) 우리가 사용한 방법들 (PCR & Southern blot법)이 분자생물학적 연구방법인 관계로 고도의 특이성 (specificity)을 지녔기 때문이며 (2) 지난 10년간 조사 대상지역인 대구·경북지역 내에서 젖소들에게 지속적으로 BLV 감염이 증가하였기 때문으로 본다. 한편 이들 BLV positive PBMC의 chromosomal DNA를 사용해 BIV의 검출을 시도한 바, lot 3C 샘플은 BLV에 100% 감염되어 있음과 동시에 그 일부가 BIV에 감염되어 있음을 PCR 방법을 통하여 확인할 수 있었다. Bovine immunodeficiency virus (BIV) which was grouped into the Lentivirinae of family Retroviridae, was known to be causing many immunodeficiency syndromes among cows. The BIV was studied worldwide during last several years for its importance in cattle industries but nothing was reported in Korea until now. Thus we initially tried to study the existence of BIV in cattle around the Daegu·Kyungpook area by PCR related molecular techniques. As a prerequisite investigation for detecting Korean-type BIV, we had focused our aim into BLV infected cows because the BLV infected cows tend to show BIV infection with 5% ranges. Hence we randomly sampled fresh bloods from 248 cows and bulls near the Daegu·Kyungpook area and collected peripheral blood monocytes (PBMC) from the sample bloods. After extracting genomic DNA from the PBMC, we subjected it to PCR and Southern blot analysis for BIV/BLV detection. Overall, 66.9% (81/121) of the cow PBMC samples turned out to be BLV positive by PCR and the result was reconfirmed by Southern blot analysis. The value was two times higher than the previously reported results of BLV infection in Korea. The significant difference was mainly due to 1) applying highly specific methods for BLV detection such as PCR 2) that BLV was continuously spreaded in the Daegu·Kyungpook area without any notice during last ten years. We also tested the BLV positive samples with the same techniques for BIV detection. And we found some BIV positives among the lot 3C samples by PCR, which had showed 100% BLV positive.

A Study on DNA Sequences and Mutation of Integrase Region of Korean-type Bovine Leukemia Virus (BLV) pol Gene

권오식,--,--,-- THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 2004 Journal of biomedical laboratory sciences Vol.10 No.1

Bovine leukemia virus (BLV) is a causative agent for lymphoma disease in cattle including cows worldwide. BLV shares similar virion structure and characteristics with other retroviruses. The pol gene of the BLV genome produced reverse transcriptase (RT) and integrase (IN) for important roles for BLV genome integration into host cell chromosomes that is known to be coded in the 3' side of the BLV pol gene (one third portion). In this study, we have sequenced 978 bp in the 3'side of the BLV pol gene from BLV 10C3 in order to determine the BLV IN region of it. And we compared it to the nucleotide sequences of an Australian BLV isolate. As a result, nucleotide sequences of the IN region of the Korean-type BLV pol gene were mutated at a rate of 3.7%. We can confirm that the typical mutations are such as Arg (AGG) → Lys(AAG), Thr (ACG) → Met (ATG), Ile (ATT) → Val (GTT), Asn (ACC) → His (CAC), Phe (TTT) → Leu (TTG) and Asn (ACC) → Asp (GAC). From the analysis of the sequencing data, we were able to determine the zinc-finger-like "HHCC" motif in the amino terminus of BLV IN, that was H-X_(3)-H-X_(25)-C-X_(2)-C. It was also found the DD35E motif in the IN catalytic domain as D-X_(56)-D-X_(35)-E. It fits very well to the consensus sequences of retroviral IN as well as HHCC motif.

A Study on DNA Sequences and Mutation of Integrase Region of Korean-type Bovine Leukemia Virus (BLV) pol Gene

Kwon, Oh-Sik,Kang, Jung-Soon,Park, Hyun-Jin,Yoo, Min The Korean Society for Biomedical Laboratory Scien 2004 Journal of biomedical laboratory sciences Vol.10 No.1

Bovine leukemia virus (BLV) is a causative agent for lymphoma disease in cattle including cows worldwide. BLV shares similar virion structure and characteristics with other retroviruses. The pol gene of the BLV genome produced reverse transcriptase (RT) and integrase (IN) for important roles for BLV genome integration into host cell chromosomes that is known to be coded in the 3' side of the BLV pol gene (one third portion). In this study, we have sequenced 978 bp in the 3' side of the BLV pol gene from BLV 10C3 in order to determine the BLV IN region of it. And we compared it to the nucleotide sequences of an Australian BLV isolate. As a result, nucleotide sequences of the IN region of the Korean-type BLV pol gene were mutated at a rate of 3.7%. We can confirm that the typical mutations are such as Arg (AGG) $\rightarrow$ Lys (AAG), Thr (ACG) $\rightarrow$ Met (ATG), Ile (ATT) $\rightarrow$ Val (GTT), Asn (ACC) $\rightarrow$ His (CAC), Phe (TTT) $\rightarrow$ Leu (TTG) and Asn (ACC) $\rightarrow$ Asp (GAC). From the analysis of the sequencing data, we were able to determine the zinc-finger-like "HHCC" motif in the amino terminus of BLV IN, that was H-$X_3$-H-$X_{25}-C-X_2$-C. It was also found the DD35E motif in the IN catalytic domain as D-$X_{56}$-D-$X_{35}$-E. It fits very well to the consensus sequences of retroviral IN as well as HHCC motif.

Survery of Seroconversion Rates against Hantavirus in Laboratory Rodents

우영대,--,--,--,-- THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 2003 Journal of biomedical laboratory sciences Vol.9 No.2

Hantavirus ale rodent-borne RNA virus that belongs to the family Bunyaviridae. Those viruses persistently infect a variety of rodents, and are transmitted by aerosols of their urine, feces and saliva. Antibody titers of sera obtained from normal laboratory rodents against hantavirus were investigated by indirect immunofluorscence antibody technique (IFA). Seroconversion rates of normal laboratory rodents showed higher in rats than that from hamster and mongolian (M), gerbil. Theses rates of normal laboratory rodents also showed higher in titers against puumala virus (PUUV) than in hantaan (HTNV) and seoul virus (SEOV). We are concerned about infections caused by hantaviruses, especially by PUUV, occurred in laboratory rodents.

Inhalation Effects of Korean Ginseng and Pine Needle on the Protection from Injury of Mouse Lung by Formaldehyde Exposure

Jung Hyuk,Kim Sang-Gi,Shin Dong-Chul,Choi Gui-Hyang,Kim Bo-Ae,Kim Sang-Kook,Kim You-Young The Korean Society for Biomedical Laboratory Scien 2005 Journal of biomedical laboratory sciences Vol.11 No.3

Formaldehyde (FA) is an important industrial chemical, but it can cause allergic reactions, sick building syndrome and so on. It has also been observed to cause cancer in scientific studies using laboratory animals, and it even causes cancer in humans. Natural products such as ginseng and pine needle containing complicated mixtures of organic chemicals are widely used in the world, because their effective components are responsible for some pharmacological activities including antioxidative effect, anticancer effect. We investigate the effect of Korean ginseng (GE), pine needle extract (PE) and combined GE and PE (cNPE) on mouse lung injury by FA exposure. GE, PE and cNPE was directly transported to pulmonary cells through respiratory organ by nebulizer inhalation. In the case of FA exposure, the pulmonary structure was damaged and its function became abnormal. However, cNPE-FA, GE-FA, and PE-FA treated groups showed similar with the control group compared with FA group. Among them, GE was proved to be more effective than any other extracts. These results demonstrate that natural product extracts could protect pulmonary structure and function against FA exposure.

열성질환 환자에서 라임병균 Borrelia burgdorferi 감염증 진단을 위한 혈청학적인 분석

김종배,김영미 THE KOREAN SOCIETY FOR BIOMEDICAL LABORATORY SCIEN 1997 Journal of biomedical laboratory sciences Vol.3 No.2

현재 라임병을 진단을 위해 국내에서 이루어지고 있는 연구 방법은 원인균인 B. burgdorferi항원에 대한 항체를 검출하는 혈청학적인 방법이다. 하지만 대부분의 경우 항원으로 사용되는 균주가 다른 나라에서 분양받은 균주이므로 이때 얻어진 결과를 유추해석하는데 어려움이 수반되고 있다. 본 실험에서는 국내 분리균주에 대한 항체를 검출하는 조건을 확립하고, 이를 토대로 하여 불명열 환자의 항체가 항원에 반응하는 양상을 파악함으로써 국내 분리균주를 이용한 혈청학적인 분석방법으로 국내 라임병 진단에 보다 적합한 조건을 제시하고자 하였다. 본 실험 결과 병원에 의뢰된 불명열 환자의 혈청이 B. burgdorferi sensu lato 중 3가지 균종의 항원에 대해 평균 약 8%정도의 양성반응을 보이는 것으로 미루어볼 때 이들 중 B. burgdorferi에 의한 열성질환 감염증이 존재할 것으로 사료된다. 특히 국내 분리균주 중 가장 많이 분리된 바 있는 B. afzelii에 대한 양성반응이 14.3%난 나타난 것으로 미루어 아직 밝혀지지 않은 국내 발생 라임병의 원인균은 주로 B. afzelii일 가능성이 가장 크다고 할 수 있을 것이다. 또한 효소결합면역 측정법(ELISA)을 이용하여 양성으로 판별된 혈청에 대해 immunoblotting법을 실시한 결과 주로 41kDa (flagellin), 27kDa, 31kDa (OspA), 34kDa (OspB)에서 band가 나타났다. 본 실험의 결과 국내 열성질환 환자들 중 라임병균에 의한 감염증이 있을 것으로 사료되므로 국내 분리균주를 항원으로 사용한 효소결합면역 측정법 (ELISA), immunoblotting법과 같은 혈청학적진단이 이루어져야 한다고 사료된다. Currently, the laboratory diagnosis for lyme disease have been performed with the detection of antibodies against Borrelia burgdorferi. However there might be some difficulties in the interpretation of obtained results due to the usage of foreign isolates as an antigen in the test. Therefore the optimization of serological tests with Korean isolates of B. burgdorferi as an antigen would be needed to establish the standardized diagnostic method for the detection of antibodies against B. burgdorferi infection. In this study, the optimization of ELISA was investigated with experimentally challenged rabbit sera and sonicated B. burgdorferi antigens of Korean isolates. Of 217 human patient's sera with unknown fever, the mean seropositivities in ELISA, done under the optimized conditions obtained in this study, were found to be about 8% against B. burgdorferi sensu lato, showing the highest seropositivity of 14.3% against B. afzelii. In immunoblotting assay with ELISA-positive human sera, the major reactive bands were 41kDa (flagellin) which might be the indication of early infection, and 27kDa, 31kDa (OspA), 34kDa (OspB) which are the characteristics of late infection. Obtained results in this study might strongly indicate the possibility of B. burgdorferi infections in Korea.

Sarcopenia and Age-Related Changes in Body Composition in Korean Older Persons

Im, Jee-Aee,Kim, Sang-Hwan,Lim, Hee-Jung,Kim, Chang-Hee,Suh, Sang-Hoon The Korean Society for Biomedical Laboratory Scien 2008 Journal of biomedical laboratory sciences Vol.14 No.1

We examined the prevalence of sarcopenia and age-related changes in body composition in Korean older persons. Community dwelling 77 men and 65 women($60{\sim}88\;yr$) were recruited for this study. Fat-free mass and the percent body fat were determined using bioelectrical impedance analysis. Isometric grip strength was measurement using grip strength dynamometer. Serum levels of fasting glucose, total cholesterol, HDL-cholesterol, and triglyceride were assayed. The prevalence of sarcopenia was found to increase with aging (men in their sixties 82.6%, seventies 96.6% and eighties 100%, and women in their sixties 47.4%, seventies 63%). In both gender groups, handgrip strength was inversely correlated with age and positively correlated with height, lean mass, and fat free mass. Better handgrip strength was related with higher weight in the men and with lower heart rate in the women. Fat free mass, age, and gender were found to be independent factors significantly associated with handgrip strength in the multivariate analysis. In summary, results of the present study suggest that handgrip strength is well associated with age, gender, and fat free mass and the prevalence of sarcopenia is increased with age and is exceeded by 40% among Korean persons older than 60 years.

Protective Effect of Mulberry Leaf and Yacon Extract Induced Hyperlipidemia in Obese Rats

Lim, Yong,Oh, Ji Hye,Park, Un Kyu,Huh, Man Kyu,Hwang, Seock-Yeon The Korean Society for Biomedical Laboratory Scien 2020 Biomedical Science Letters Vol.26 No.2

There has been a lot of interest in medicinal effects for hyperlipidemia from the natural product since the therapy of atherosclerosis has emerged as social concern. Rats were acclimated to the feeding environment for one week and induced to obesity with high fat diet during 4 weeks until their body weight were more than 30% of normal range. To evaluate the effect of hyperlipidemia of mulberry leaf of Morus alba and yacon tuber of Smallanthus sonchifolia extracts (MLYT), 1%, 3%, 5% of MLYT were treated to Sprague-Dawley rats. Aspartate transaminase (AST) was significantly increased 26.2% in high fat diet group (G2) compared to normal diet group (G1) (P<0.05). But AST were significantly decreased in high fat diet with 0.5% hydroxycitric acid diet group (G3) and all the MLYT treated groups compared to G2. Total cholesterol, low-density lipoprotein (LDL), triglycerides and phospholipids were observed significant improvements in the MLYT-treated groups (P<0.05). These improvements in liver and feces were further supported by the lipid content. MLYT remarkably suppressed the level of lipid peroxidation caused by high-fat diet in rats. The level of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase activity. As a result, the proper combination of mulberry leaves and yacon tuber extract will have a synergistic effect on hyperlipidemia. These results indicate that MLYT could be a candidate for the improvement of hyperlipidemia.

Effect of Extraction Ethanol Concentration on Antioxidant and Anti-Inflammatory Activity of 30-Year-Old and 120-Year-Old Dangyuja (Citrus maxima (Burm.) Merr.)

Lee, Sung-Gyu,Lee, Dongsup,Kang, Hyun The Korean Society for Biomedical Laboratory Scien 2020 Biomedical Science Letters Vol.26 No.2

Dangyuja (Citrus maxima (Burm.) Merr.) is a native fruit of great economic importance in Jeju island in Korea. To provide experimental evidence for the antioxidant and anti-inflammation properties on extraction ethanol concentration of Dangyuja, 2 cultivars, including 30-year-old and 120-year-old were evaluated. 30-year-old Dangyuja 50%, 70% ethanol extracts had the highest polyphenol and flavonoid content, and the strongest 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity. To investigate the anti-inflammatory activity of Dangyuja ethanol extracts, we used BV-2 microglia cells and induced inflammation using lipopolysaccharide (LPS). Then, we measured levels of inflammatory mediators as nitric oxide (NO). Among the 6 extracts, 30-year-old Dangyuja 50% ethanol extracts show the highest anti-inflammatory activity. The results suggest that 30-year-old Dangyuja 50% ethanol extracts provides significant health benefits and may be used for developing new functional materials.