포도 VIASR 유전자 프로모터의 분리 및 발현 분석

길준영,피재호,Kihl, Joon-Yeong,Pyee, Jae-Ho 한국생명과학회 2007 생명과학회지 Vol.17 No.8

VvMSA, a grapevine ASR which is highly inducible by sugar and abscisic acid signals was previously shown to be a transcription factor for a hexose transporter gene VvHT1. We isolated a cDNA clone, VlASR which is regulated temporally during the grape berry development by ACP RT-PCR (annealing control primer reverse transcriptase-polymerase chain reaction) and it proved identical to VvMSA. RT-PCR and real-time PCR analyses revealed that the VlASR gene was expressed in berries at fruit set and that its expression increased as berries aged but decreased at the late ripening stage. In order to understand the regulatory mechanism of the asr gene, a genomic fragment was cloned from grapevine. The genomic DNA was 1375 bp long and a sugar box (sucrose box 3 and sucrose responsive element 1) was identified in the 611 bp upstream region of the open reading frame. Analysis of the VlASR promoter::reporter gene fusion demonstrated that this promoter was expressed in transgenic Arabidopsis even without sucrose treatment. This result suggests that the ASR/VvHT1-mediated sugar/ABA signaling, previously reported in grapevine, may not function in Arabidopsis which has no ASR homologue. 포도 ASR (VvMSA) 단백질은 hexose transporter 유전자 VvHT1의 전사를 조절하는 조절 인자 중의 하나로서 sugar 및 abscisic acid (ABA) 신호에 의해 발현이 유도된다. 본 연구진은 ACP RT-PCR (annealing control primer reverse transcriptase-polymerase chain reaction) 방법을 이용하여 포도 과실발달 과정에서 조절되는 유전자 중 VvMSA와 동일한 cDNA (VlASR)를 클로닝하였다. 이 유전자는 착과 시기에 발현되기 시작하여 과실이 발달하면서 점점 증가하여 착과 후 10 주에 가장 많이 발현되며, 숙기 후반에는 도리어 발현양이 감소하였다. 포도 asr 유전자의 조절기작을 밝히기 위해, 이 유전자의 genomic clone을 분리하였다. 총 1375 bp로 이루어진 이 유전자 절편에는 open reading frame과 100 bp의 intron을 포함하고 있다. 약 600 bp 길이의 프로모터 내에는 sugar 신호전달과 연관이 있는 것으로 알려진 sugar box(sucrose box 3 +sucrose response box 1)가 있다. 프로모터 절편을 reporter 유전자와 연결하여 Arabidopsis에 도입하고 형질전환체를 분석한 결과, reporter 유전자는 sucrose 처리와 상관없이 항상 발현되었다. 이러한 결과는 포도에서 보고된 ASR/VvHT1를 매개로 하는 sugar/ABA 신호전달계가 asr 유전자가 없는 Arabidopsis에서는 작동되지 않음을 시사하고 있다.

Cyclosporine에 혈액학적 반응을 보인 Refractory Anemia 1예

길준영,윤환중,전의건,오덕연,김삼용,김종완,박종우 大韓血液學會 1992 大韓血液學會誌 Vol.27 No.2

저자들은 refractory anemia로 진단받고 23개월간 지속적인 적혈구 수혈(매월 2units씩 총 47units)을 필요로 했던 58세 여자 환자에 cyclosporine을 투여하여 21개월간 혈액학적 반응 을 경험하였고 이 기간에는 수혈이 필요치 않았다. Refractory anemia의 발병기전은 아직 분명치 않으나 일부 환자에선 면역억제제에 반응함으로써 일부 면역기전이 작용한다고 생각된다. The myelodysplastic syndromes which encompass refractory anemia are characterized by ineffective hematopoiesis, refractory cytopenias, and an increased risk of leukemic transformation. No currently available treatment has been shown to be consistently effective in producing sustained improvement in hematopoiesis or in delaying leukemic evolution. Cyclosporine has been used for the therapy of patients with aplastic anemia. We report a case of refractory anemia who responded to cyclosporine(Sandimun®). A 58-year-old woman was admitted to the Chungnam National University Hospital for the evaluation of pancytopenia. Blood examination showed 2,600/㎣ of white blood cells, 5.9g/dL of hemoglobin, and 38,000/㎣ of platelets. Reticulocyte index was 0.2%. The levels of serum iron, iron-binding capacity, vitamin B_l2, and folic acid were in normal range. Bone marrow biopsy revealed hypercellularity, dysmyelopoiesis, dyserythropoiesis, myeloid hyperplasia. The hematologic findings were compatible with the diagnosis of refractory anemia and the diagnosis of aplastic anemia could be excluded. Treatment with oxymetholone(50㎎/day) and pyridoxine(300㎎/day) was started. Packed red cells transfusions(total of 47 units) were required over a period of 23 months. An oral cyclosporine(Sandimun®) therapy was started at a dosage of 300㎎ per day. One month after cyclosporine therapy hemoglobin level began to increase and reached 11.6g/dL after 12 weeks. The dose of cyclosporine was reduced to 200㎎ per day and discontinued after total of 19 weeks of therapy. Her hemoglobin level remained stable between 13 and 15g/dL without transfusion. After a 21 months of hematological remission, she was readmitted because of progressive dyspnea after undergoing a cataract operation 2 months ago. Despite transfusion of packed red cells and plateletpheresis to which she was refractory. Her hemoglobin level was around 3g/dL and platelet count decreased progressively. Retrial of cyclosporine(900㎎/day for 2 weeks) and a trial of GM-CSF(400㎍/day for 7 days) were of no benefit. She succummed to gastrointestinal and intraperitoneal bleeding. The exact mechanism of cyclosporine in this case is uncles but it is reasonable to ascribe the striking hematologic improvement to cyclosporine. As some reports showed responses to immunosuppressive therapy in refractory anemias, in some proportion of patients with refractory anemia, immune mechanism may be involved in the pathogenesis. The role of immunosuppressive therapy in refractory anemia should be further clarified.

재생불량성 빈혈의 임상적 고찰

길준영 ( Gil Jun Yeong ),전의건 ( Jeon Ui Geon ),윤환중 ( Yun Hwan Jung ),김백수 ( Kim Baeg Su ),최용석 ( Choe Yong Seog ),조덕연 ( Jo Deog Yeon ),김삼용 ( Kim Sam Yong ) 대한내과학회 1993 대한내과학회지 Vol.44 No.3

연구배경 : 재생불량성 빈혈은 우리나라를 비롯하여 극동지역에서 호발하는 중요한 질환이며 우리나라 재생불량성빈혈 환자의 임상상은 서양과 차이가 있는 것으로 알려져 있다. 재생불량성 빈혈 환자를 임상적으로 관찰하여 생존기간과 예후인자등을 알아보고자 1985년 8월부터 1991년 7월까지 6년동안 충남대학교병원 내과에서 입원가료를 받은 재생불량성 빈혈 환자 32예에 대하여 분석하였다. 방법 : 모든 대상환자에 일차적으로 oxymetholone(50~100mg/d)을 경구 투여하였고 필요에 따라 prednisolone (5~10 mg/d)을 추가하였다. Androgen 요법에 반응이 없는 20예중 9예에 antilymphocyte globulin (20mg/kg/d)을 4일간 정맥주사 하였으며 동시에 methylprednisolone (20mg/kg/d)을 5일간 정맥주사 하였다. 결과 : 1) 연령별로는 21~30세가 12예 (37.5%)로서 가장 많았으며 40세 이하가 25예(78.2%)였다. 2) 원인을 추정할 수 없는 경우가 26예(81.2%)로 대부분을 차지하였고 원인을 추정할 수 있는 경우가 6예 (18.8%)였는데 한약제 2예(6.3%), benzene 2예 (6.3%), 항진균제 1예(3.1%), 급성간염 B형 1예(3.1%) 였다. 3) Androgen을 중심으로 한 치료에 31예 중 5예(16.1%)에서 완전방응을, 6예(19.4%)에서 부분반응을 보여 반응율은 35.5%이였다. Androgen 치료에 반응이 없었던 20예중 9예에 2차적으로 시행한 antilymphocyte globulin 면역조절요법에 2예(22.2%)에서 완전방응을 보여 전체 42%의 반응율을 보였다. 4) 초진시 중증 재생불량성 빈혈 환자 20예(64.5%)의 72개월 생존율은 79.3%, 중앙생존기간은 32(2~72+)개월이었다. 중등도의 재생불량성 빈혈 11예(37.5%)의 72개월 생존율은 90%, 중앙생존기간은 37(13~72+)개월이었다. 50 남자 환자에서 여자 환자보다 반응율이 더 높았고 (61.5% vs 27.8%, p<0.05). 원인추정이 가능했던 예에서 원인을 알수 없었던 예보다 더 높은 반응율을 보였다(66.7% vs 28%, p<0.05). 그러나 초진시의 혈소판수, 과립구수, 또는 교정 망상적혈구수는 치료반응에 영향을 미치지 못하였다(p>0.05). 결론 : 재생불량성 빈혈 환자 32예중 31예에서 androgendmf 중심으로한 고식적치료와 면역억제요법만으로도 72개월 생존율이 83.1%였으며 서양환자에서의 예후보다 훨씬 양호한 것으로 판단된다. 따라서 골수이식은 초진시 중증 재생불량성 빈혈 진단을 받은 환자 중에에서도 고위험군을 선정하여 시행하는 것이 바람직하다고 생각되며 중등도 이하의 재생불량성 빈혈에서는 면역조절요법(항림프구 혈청 치료)과 고식적치료가 우선적으로 권장되어야 할 것으로 사료된다. Background : It is well konwn that aplastic anemia is more prevalent in the Far East than elsewhere in the world. There have been many suggestions that the clinical features of the patients with aplastic anemia in Korea would be somewhat different from that of western countries. Analysis of clinical data of 32 cases of aplastic anemia diagnosed at Chungnam National University Hospital from August 1985 to July 1991 was done. Methods : Initially all patients were treated with oxymetholone (50-100mg/d) with or without prednisolone (5-10mg/d) and supportive care. The patients who did not respond to androgen therapy were treated with antilymphocyte globulin (ALG) (20mg/kg/d for 4 days) plus methylprednisolne (methyl PD) (20mg/kg/d for 5 days). Analysis of reponse to therapy was done in 31 patients who could be followed for more than 3 months. Results : 12 patients (37.5%) were in their third decade and 25 cases of patients (78.2%) were below 40 years of age. The incidence decreased in older age groups. Exposure to possible toxic agents were seen in 6 cases (18.8%) ; benzene was counted in 2 cases (6.3%), herb drug in 2 cases (6.3%), antifungal agent in 1 case (3.1%). One case of aplastic anemia occured after acute viral hepatitis infection (type B) (3.1%), Eleven (35.5%) out of thity-one patients responded to androgen therapy (CR : 16.1%, PR : 19.4%). Nine out of the twenty patients who did not respond to androgen therapy were treated with ALG plus methyl PD. Two patients (22.2%) out of 9 showed complete responses. Overall response of present series of patients was 42% (oxymetholone±prednisolone therapy : 35.5%, ALG+methyl PD : 22.2%). By Kaplan-Meier product limit estimation, median survival time of 31cases was 36 months and the actuarial survival at 72 months was 83.1%. The median survival time of 31 cases was 36 months and the actuarial survival at 72 months was 83.1%. The median survival time of severe aplastic anemia (32 months) was similiar with that of moderately severe aplastic anemia (37 months). Factors associated with favourable survival were male sex and presence of etiological factor (p<0.05). Conclusions : In the present series, the survival rate of patients with aplastic anemia were somewhat different from that of western countries. Since the prognosis of patients with aplastic anemia in Korea is more favourable. A randomized controlled study would be needed to define the therapeutic role of bone marrow transplantation in patients with severe aplastic anemia in Korea.

자외선 Hormesis가 딸기의 페놀류 함량에 미치는 영향

조용진,김철진,김종태,김태은,배기석,길준영,피재호,이상국 한국산업식품공학회 2008 산업 식품공학 Vol.12 No.3

재생불량성 빈혈의 임상적 고찰

김삼용,길준영,전의건 충남대학교 의과대학 지역사회의학연구소 1991 충남의대잡지 Vol.18 No.2

A retrospective analysis of clinical data of 32 cases of aplastic anemia diagnosed at Chungnam National University Hospital from August 1985 to July 1991 was done. 1) The highest incidence of aplastic anemia was seen in the third decade and 25 cases of patients (78.2%) were below 40 years of age. The incidence decreased in older age groups. 2) Exposure to possible toxic agents were seen in 6 cases(18.8%), among which benzene was related in 2 cases(6.3 % ), herb drug in 2 cases(6.3 % ), acute viral hepatitis(type B) in 1 case(3.1 % ), antifungal agent in 1 case(3.1 % ). 3) Initially all patients were treated with oxymetholone with or without prednisolone and supportive care. Eleven(37.9 %) out of twenty-nine patients showed responses(CR: 17.2%, PR: 20.6%). Nine out of the eighteen patiens who did not respond to androgen therapy were treated with antilymphocyte globulin (ALG) plus methylprednisolone. Two patients(22.2 %) out of 9 showed complete responses. 4) Overall responses of present series of patients was 44.8 % (androgen± prenisolone therapy: 37.9%, ALG+ methylprednisolone: 6.9%). The actuarial survival of all patients at 72 months was 83.4%. The median survical rate of severe aplastic anemia(32 months) was similiar to that of moderate aplastic anemia(37 months). 5) Factors associated with favourable survival were male sex and presence of etiological factor. Since the prognosis of aplastic anemia in Korea is quite favorable, randomized controlled study would be needed to define the therapeutic roles of bone marrow transplantation of immunosuppressive therapy in patients with severe aplastic anemia in Korea.

백혈병 세포주에 대한 Biological Response Modifiers의 항암제 세포독성 증강효과

이윤영,전의건,길준영,윤일국,유관희,안병준,김삼용 충남대학교 약학대학 의약품개발연구소 1995 藥學論文集 Vol.11 No.-

To investigate the possibility of increased cytotoxicity of anticancer drugs, we treated human leukemia cells with combinations of anticancer drugs and biological response modifiers(BRMs). Using the colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay, we evaluated the chemosensitivity of 8 anticancer drugs (vincristine, vinblastine, adriamycin, cisplatin, etoposide, cytosine arabinoside, bleomycin, and cyclophosphamide), and the anticancer effects of 3 BRMs(interleukin-2, alpha interferon, and gamma interferon) combined with these anticancer drugs against human leukemia HL-60 and KG-l cells. The results were as follows; in the chemosensitivity of 8 anticancer drugs, VCR, VBL, ADR, and CPDD were effective, while VP-l6, ara-C, Bleo, and CYC produced less than 50% inhibition of HL-60 and KG-l cells lines. Among 3 BRMs investigated, all of them showed less than 20% inhibition of KG-l cell lines and none were effective against HL-60 cells. In the anticancer effects of 3 BRMs, all of them showed about 20% inhibitory effects against KG-l cells, but there were not any effects against HL-60 cells. All of the anticancer drugs markedly increased cytotoxic effects when they were combined with BRM. Especially, the ID_50 values of VCR, VBL and ADR when combined with BRMs decreased to 67~3%. These results demonstrate that some BRMs can markedly increase the cytotoxicity of VCR, VBL, ADR, CPDD, VP-l6, ara-C, Bleo, and CYC and suggest possible clinical usefulwess.

진행암 환자에서 Cisplatin 병용화학요법시 Ondansetron의 오심 구토 조절 효과

전의건,윤환중,길준영,조덕연,김삼용 최신의학사 1992 最新醫學 Vol.35 No.12

구인두 캔디다증에 대한 Fluconazole의 치료효과

전의건,윤환중,길준영,조덕연,김삼용 최신의학사 1993 最新醫學 Vol.36 No.5

Genetic and Physiological Discrepancies from Isolates of Sclerotinia homoeocarpa causing Zoysiagrass Dollar Spot Disease

박대섭,김경덕,길준영,피재호,Park, Dae-Sup,Kim, Kyung-Duck,Kihl, Joon-Yeong,Pyee, Jae-Ho Turfgrass Society of Korea 2006 한국잔디학회지 Vol.20 No.1

난지형 잔디인 한국 안양잔디에서 달라스팟의 병원균인 Sclerotinia honoeocarpa의 isolate, Scz1이 최근 새롭게 동정되었다. Scz1은 한지형 잔디인 크리핑 벤트그래스에서 분리된 표준 균주인 Scb1과는 다른 균사의 색상, 균사간의 친밀도 그리고 병 기주 특이성을 가지는 것으로 알려졌다. 본 연구에서는, Scz1, Scz2(난지형 잔디에서 분리한 또 다른 달라스팟 병원균) 그리고 Scb1을 분자생물학적인 연구, internal transcribed spacer(ITS) 와 random amplified polymorphic DNA(RAPD) assays를 이용하여 동정 및 유전자적 차이를 알아보았다. ITS 실험의 결과, 3개의 isolates가 ITS 부분적 염기 서열 비교 BLAST에 등록되어 있는 S. homoeocarpa의 ITS 염기 서열과 $94{\sim}97%$의 동일성을 지니는 것으로 밝혀졌다. RAPD 실험 결과로는, Scz1과 Scb1의 similarity matrix 범위는 0.167이였고, Scz2와 Scb1은 0.139 그리고, Scz1과 Scz2은 0.713이였다. 계통수(系統樹) 결과는 Scb1과는 달리 Scz1과 Scz2는 유전적으로 높은 동일성을 지니고 있어, 같은 분류에 속한다는 것을 알 수 있었다. 달라스팟 병원균 억제에 효과적인 농약인 프로피코나졸에 대한 $EC_{50}$은 Scz1은 0.012 ${\mu}g/ml$, Scz2은 0.003 ${\mu}g/ml$ 그리고 Scb1은 0.030 ${\mu}g/ml$이었다. 상기 결과로, 동일 병원 기주성과 유사한 유전적 친밀성을 보인 Scz1과 Scz2는 S. homoeocarpa의 동일 그룹에 속하였으나 농약 민감도에서는 차이점을 보였다는 것을 알 수 있었다. 향후, 보다 더 많은 한지형과 난지형 잔디에서 분리된 병원균들을 이용하여 유전적 다양성을 밝히는 연구가 진행되어야 할 것이다. Scz1, an isolate of Sclerotinia homoeocarpa, was recently reported as a novel pathogen responsible for dollar spot disease in Zoysiagrass, a warm season turfgrass. Scz1 possessed different characteristics on mycelial pigment, mycelial affinity and host pathogenecity compared to those of Scb1, a typical isolate, obtained from creeping bentgrass, a cool season turfgrass. In this study, only three isolates, Scz1, Scz2(another analogous isolate of Sclerotinia homoeocarpa from zoysiagrass), and Scb1, were examined at the molecular level using the internal transcribed spacer(ITS) and random amplified polymorphic DNA(RAPD) assays to verify their identification and genetic variation. As a result of ITS assay, partial ITS sequences of three isolates showed 94-97% similarity with a standardized ITS sequence of S. homoeocarpa registered on BLAST. In the analysis of RAPD, range value through similarity matrix was 0.167 between Scz1 and Scb1, 0.139 between Scz2 and Scb1, and 0.713 between Scz1 and Scz2, respectively. Furthermore, tendegram analysis indicated that Scz1 and Scz2, unlike Scb1, were clustered together as accompanying a high genetic similarity. In in vitro fungicide bioassay, $EC_{50}$ value representing the sensitivity degree to propiconazole, a well-known fungicide for dollar spot disease, was 0.012 ${\mu}g/ml$ for Sczl, 0.003 ${\mu}g/ml$ for Scz2, and 0.030 ${\mu}g/ml$ for Scb1. From all data taken, we concluded that both Scz1 and Scz2 belonged to one group of S. homoeocarpa, since they exhibit the same host range and high level of genetic similarity, whereas their chemical competences to a fungicide were different. This study would provide further approach for assessing genetic diversity of S. homoeocarpa isolates as well as characterizing individual isolate against chemical exposure.

Neisseria gonorrhoeae 분리균의 항생제 내성 양상 분석

성원근,정경태,길준영,김석하,오희복 대한감염학회 2001 감염 Vol.33 No.5

Background : The aim of this study was to monitor trends in antimicrobial susceptibilities of Nesseria gonorrhoeae isolates, in particular, to examine the possibility of increasing prevalence of fluoroquinolone resistance in Korea and the relationship between patterns of mutations involving gyrA and parC genes and the ciprofloxacin resistance level. Methods : The antimicrobial susceptibilities of 489 gonoccocal isolates which were nationwide collected from patients visiting Health Centers were determined by NCCLS disk diffusion and agar plate dilution methods. PCR and direct DNA sequencing of the amplicons were performed to identify mutations within the quinolone resistance-determining regions of gyrA and parC genes. Results : The proportion of resistance to penicillin, tetracycline, or both remained as high as 94%. The isolates with ciprofloxacin resistance remarkably increased from 1% in 1999 to 5% in 2000 and also resistance to cefoxitine and ceftriaxone were shown to be increased. The strains resistant to spectinomycin was little reported. Four isolates with 16 ㎍/mL of MIC for ciprofloxacin all showed the same alternations of Ser-91 to Phe, Asp-95 to Gly in GyrA and Ser-87 to Arg in ParC, but ciprofloxacin-susceptible strains have little amino acid substitution. Conclusion : Considering the increasing prevalence of isolates with resistance to ciprofloxacin and ofloxacin, it is likely that the antibiotics such as spectinomycin, or ceftriaxone are recommended as the first-line treatment for gonoccocal infections in Korea. The results from this study suggest that mutation analysis for quinolone resistance-determining regions of gyrA and parC genes are important in epidemiological studies for the spread of ciprofloxacin resistant strains.(Korean J Infect Dis 33:338∼345, 2001)