Functional Analysis of the First Mannosyltransferase (PIG-M) involved in Glycosylphosphatidylinositol Synthesis in Plasmodium falciparum

Youn Uck Kim,홍영진 한국분자세포생물학회 2007 Molecules and cells Vol.24 No.2

Poster Session 1 : Metabolism and Enzymology Protein Structure and Function ; Functional analysis of 1st mannose transferase for synthesizing a glycosylphosphatidylinositol in malaria and yeast PIG-M

( Youn Uck Kim ),( Yeong Jin Hong ),( Kinoshita Taroh ) 한국생화학분자생물학회 (구 한국생화학회) 2004 생화학분자생물학회 춘계학술발표논문집 Vol.2004 No.-

Characterization of Lipopolysaccharide Responsive Proteins that Binds to the 5'Flanking Regions of Mouse Rantes

Youn-Uck Kim,Youn-Hwoan Kim,Duek-Jun An,Hyuk-Chu Kwon 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.1

When macrophage (like the RAW264.7 cell line) was stimulated with lipopolysaccharide (LPS), factors that bind specifically to the LPS responsive element (LRE) of murine Rantes gene appeared in the nucleus. An electrophoretic mobility shift assay (EMSA) detected 2 specific bands, designated as S (slow) and M (middle). The S band appeared within 15 min of LPS stimulation, and reached its highest intensity within 2 h. The M band was present in unstimulated cells, but after stimulation its intensity increased and reached its highest intensity also in about 2 h. Significantly, in LPS hyporesponsive 10-9 macrophage like cells, the S band was absent. The M band was present in equal amounts in stimulated and unstimulated cells. The results suggest that the S band was induced by LPS stimulation. In the nuclear extract, the native molecular weight of the S band-forming factor was approximately 270 kDa, and that of the M bands-forming factor was approximately 140 kDa. U.V. cross linking studies consistently showed at least 2 different polypeptides of approximate molecular mass of 70 kDa, both in the S band-forming factor (complex) and the M band-forming factor (complex). In the nuclear extracts of both the LPS stimulated and unstimulated cells, we detected a factor with approximate molecular mass of 120 kDa that could convert the S band-forming complex to the M bandforming complex. This factor, designated as a converting factor, is a protein phosphatase since its activity was blocked by okadaic acid, an inhibitor of Ser/Thr protein phosphatase. Also, purified protein phosphatase type 1 (PP-1) could convert the S bandforming complex to the M band-forming complex.

Functional analysis of the first mannosyltransferase (PIG-M) involved in glycosylphosphatidylinositol synthesis in Plasmodium falciparum.

Kim, Youn Uck,Hong, Yeongjin Korean Society for Molecular Biology 2007 Molecules and cells Vol.24 No.2

<P>The mammalian glycosylphosphatidylinositol (GPI) anchor consists of three mannoses attached to acylated GlcN-(acyl)PI to form Man(3)-GlcN-(acyl)PI. The first of the three mannose groups is attached to an intermediate to generate Man-GlcN-(acyl)PI by the first mannosyltransferase (GPI-MT-I). Mammalian and protozoan GPI-MT-I have different substrate specificities. PIG-M encodes the mammalial GPI-MT-I which has 423 amino acids and multiple transmembrane domains. In this work we cloned PIG-M homologues from humans, Plasmodium falciparum (PfPIG-M), and Saccharomyces cerevisiae (GPI14), to test whether they could complement GPI-MT-I-deficient mammalian cells, since this biosynthetic step is likely to be a good target for selective screening of inhibitors against many pathogenic organisms. PfPIG-M partially restored cell surface expression of the GPI-anchored protein CD59 in PIG-M deficient mammalian cells, and first mannose transfer activity in vitro; however, this was not the case for GPI14.</P>

A Ser/Thr Specific Protein Kinase Activates the Mouse Rantes Gene after Lipolpolysaccharide STimulation

Kim, Youn-Uck,Kim, Youn-Hwoan,An, Duek -Jun,Kwon, Hyuk-Chu The Microbiological Society of Korea 2001 The journal of microbiology Vol.39 No.4

Macrophages stimulated by lipopolysaccharide(LPS) from gram negative bacteria undergo activation of a group of immediate early genes including Rantes. The mouse Rantes gene promoter region contains an LPS rsponsive element(LPE) We detected 3 specific bands termed B1, B2 and 3 formed by the interaction of the LPE and proteins found in LPS-stimulated RAW 367.7 cells. An additional band B4 was determined to be an Ap-1 binding protein. The B1 band appears within 1 hour of LPS nuclear extracts from LPS-stimulation, and this protein kinase enhances B1 and formation. The B1 band can be converted to band B2/B3 by adding specific heparin column fraction purified Ser/Thr specific protein phosphatases PP-1 and PP-2A can stimulate the same conversion to about the same extent. Thus, the formation of the LRE sequence binding complex appears to be regulated by Ser/Thr protein kinase and one or more Ser/Thr specific phosphatases. At least four proteins are involved in the trgulation of the LRE-dependent Rants experssion: two binding factors that bind directly to the target sequences. and two factors that control their binding. The future purification and characterization of these binding pro-teins will reveal in detail the mechanism of Rantes gene activation after LPS stimulation.

Mouse Rantes Gene Regulation

Kim, Youn Uck 선문대학교 자연과학대학 1999 자연과학대학 논문집 Vol.2 No.-

Mouse Rantes 유전자의 promoter 에는 LPS에 반응하는 염기배열이 포함되어있다. 우리는 이들 염기에 특이적으로 결합하는 인자 3종류를 확인 하였다. 이름하여 B1 B2, B3 이라 명명하였다. 이들 인자는 RAW 264.7 세포를 LPS로 자극하였을 때 Rantes 유전자의 LPS 반응염기 배열에 결합하는 성격을 가지고 있었다. 우리는 LPS의 자극이 대식세포내 의 단백질인산화 효소의 활성화를 초래 한다는 사실을 확인했으며, 이것이 Rantes 의 promoter에 결합하여 전사조절 인자에 영향을 주는 것을 확인하였다. The mouse Rantes gene promoter region contains an LPS responsive element (LRE). We detected 3 specific bands, termed Bl, B2, and B3, formed by the interaction of the LRE and proteins found in LPS-stimulated RAW 264.7 cells. We have determined that a Ser/Thr specific protein kinase is activated by LPS stimulation, and this protein kinase enhances Bl band formation. The Bl band can be converted to band B2/B3 by another specific heparin column fraction. Purified Ser/The specific protein phosphatases PP-1 and PP-2A can stimulate the same conversion to about the same extent. Thus, the formation of the LRE sequence binding complex appears to be regulated by a protein kinase and one or more Ser/Thr specific phosphatases.

세균에 의한 염증발현 기작의 규명

Kim, Youn Uck 선문대학교 자연과학대학 1999 자연과학대학 논문집 Vol.2 No.-

대식세포는 여러 가지 물질에 의하여 자극을 받는다. 우리는 Gram (-)세균 세포벽성분인 LPS의 영향을 조사하였다. 대식세포는 LPS 자극에 대응하여 Rantes를 비롯한 초기발현유전자를 발현시킨다. 이러한 발현물질은 병원 미생물대한 숙주의 신체 방어 기작에서 대단히 중요한 역할을 한다. 우리는 이미 LPS에 대응하는 초기발현유전자들은 그들의 promoter가 대단히 중요한 역할을 한다는 것을 밝혀냈으며, 이들 유전자의 공통점은 정해진 유전자 배열이 그들의 promoter부분에 존재한다는 것을 확인하였다. 이것을 LRE라 명칭하였으며, 이들 염기 배열 속에는 AP-I, ISRE, 둥 유전자 발현에 필수적인 염기 배열 등이 존재하는 것을 확인 할 수 있었다. 이 보고서에서는 이들 promoter에서는 과연 어떤 단백질이 어떻게 작용하는가를 조사하기 위하여 EMSA를 실시하였고, 좀더 정확한 정보를 얻고자하여 대식세포의 핵단백질을 부분 정제하여 이들 단백질의 성격을 파악하였다. 이들실험 결과에의하면 염기배열 -100부터 -200사이에 두 종류의 단백질이 인산화효소, 탈인산화효소에 조절되며 분자량은 약 50-55kDa인 것 이 확인 되었다. Macrophage can be stimulated with various agents, among which we studied the effects of LPS from gram negative bacteria. The various changes of LPS stimulated macrophage activates a group of immediate early gene including Rantes in mouse. It would probably play an important role in the first defence to a pathogen. We have Previously showed that murine Rantes was activated in immadiate early manner. And LPS responsive elements (LRE) in the mouse Rantes consisted of two motifs, TCAYR, which was an AP-1 half site with two flanking bases, and (A/T)(G/C)NTTYC(A/T)NTTY, which resemble in part the interferon- stimulated responsive element (ISRE). These two motifs are separated by 10 nonconsensus nucleotides. In this paper, we have futher characterized and identified LPS responding element (containing two motifs) binding factors by electrophoretic mobility shift assay (EMSA) with crude or fractionated nuclear extracts in macrophge like cell RAW 264.7. We identified that at least four proteins are involved in recognizing the LRE and form a complex : two proteins bind directly to the target sequences, and two factors do not bind directely but control the binding factors to bind the target sequences. We have determined that two DNA binding proteins are about 50kDa ; 50kDa bind to 5'-terminal (-192 to -158) which included AP-1 (6/7) sequences and 55kDa to 3-terminal region (-160 to -118) which contained T-rich sequences, repectively.

공인 인증서 검증 프로토콜 SCVP의 구현 방안에 관한 연구

안연정(Youn Jung Ahn),백선욱(Seon Uck Paek) 상명대학교 공학기술연구소 2003 공학기술연구 Vol.2003 No.2

In this paper, the implementation result of SCVP(Simple Certificate Verification Protocol) is described. The previous verification protocol, OCSP, can verify only the state of a certificate and cannot verify certification path or certification policies. We developed SCVP on OpenSSL-0.9.7 and interworked it with OCSP, thus the proposed system can verify the state of the certificate, certification path and certification policy.

Rantes 유전자의 Promoter 해석

김연욱 선문대학교 자연과학대학 2000 자연과학논총 Vol.3 No.-

대식 세포는 그람 음성 세균의 세포벽성분인 LPS에 의해 자극 유도되어 여러 가지 물길올 만들어낸다. 그중에서 chemokine이라 불리는 Rantes 가 발현되는 데 이것은 신체가 외부의 세균에 대해 방어적 기능을 발휘하는데 중요한 물질로 알려져 있다. 또한 이 Rantes는 에이즈 바이러스가 감염될 때 이유전자의 수용체를 통해서 감염된다는 것이 알려짐으로 해서 AIDS 치료 및 예방과 관련하여 관심의 대상이 되고 있다. 1040 bp로 구성된 mouse Rantes의 5' promoter에는 NF-κB 나 AP-1등 유전자의 발현을 조절하는 다수의 염기배열이 존재한다는것도 확인되었다. 또한 최근에는 지질다당체에 반응해서 역시 유전자 발현을 조절하는 새로운 LRE motif (-192/ -112)라는 것이 확인되었다. 본 연구에서는 NF-κB 과 AP-1등과 유사한 염기배열을 가진 LRE motif 를 본래의 NF-κB과 AP-1 염기배열을 인위적으로 제작하여 이들을 시험관에서 경쟁적저해를 시킴으로해서 이들의 결합을 확인하였으며. 또한 돌연변이된 염기배열올 가지고 이두가지 인자의 결합을 확인하였다. To understanding the molecular mechanisms of LPS (lipopoly-saccharide)-induced gene activation, we investigated the nature of mouse Rantes gene that can be activated by LPS with early manner without protein synthesis. Current studies showed that LPS responsive element (LRE) sequences were positioned from -192 to -112 of Rantes promoter. And an AP-I site and NF-κB site were found in that sequences. In the present study, we have characterized proteins binding to the mouse Rantes LRE region using nuclear extract of RAW 264,7 cells. Electrophoretic mobility shift assay (EMSA) was used to characterize the specific binding protein with the mutated target sequence or cold probe. And we have known that AP-I and T-rich region binding factors bound to 3' and 5'-terminal region of the target sequence.

Ilizarov 치료의 임상적 연구 : The Preliminary Reports

서광윤,권칠수,김용욱,김형수 인제대학교 1991 仁濟醫學 Vol.12 No.4 sup