사상성진균인 Cryphonectria parasitica에서의 Stomatin-like protein (CpSlp1)의 기능분석

이재현 전북대학교 일반대학원 2023 국내석사

The chestnut blight fungus, Cryphonectria Parasitica, which interacts with Cryphonectria Hypovirus 1 (CHV1), is known as a useful model for studying fungul-virul interaction mechanisms. In our previous study, the sectorization was observed in two gene (CpBckl, CpSlt2) mutants associated with cell wall integrity pathways. As a characteristic of sectorization, it decreased compared to the wild type (EP155/2), while growth recovered but did not development. In addition, 73 genes were found to be common differentially expressed genes (DEGs) between the sectored progenies and the corresponding parental mutant strains (CpBck1-sectored progeny vs CpBck1-null mutant and the CpSlt2-sectored progeny vs CpSlt2-null mutant) via RNA sequencing analysis. Of the 73 DEGs, 22 DEGs were found to be affected by hypovirus CHV1 infection. In this study, a gene encoding Stomatin-like protein (CpSlp1) was selected among those genes affected by both CHV1 and sectorization. BLAST search revealed that the cloned gene showed high homology with one of membrane protein family of Coniella lustricola. To analyze the biological function of the putative Stomatin-like protein of Cryphonectria parasitica, we constructed CpSlp1-null mutant via transformation based on homologous recombination. Of the 192 transformants, one CpSlp1-null mutant was finally obtained using PCR screening. The phenotype of CpSlp1-null mutant was similar to the wild-type strain, EP155/2, on PDAmb but CpSlp1-null mutant ‘s growth rate was lower than EP155/2. No differences in responsiveness was observed to various stressors such as ROS, and osmotic stress. However, the growth rate of CpSlp1-null mutant was lower than EP155/2 in the PDAmb medium containing SDS, a cell wall disturbing agent. To confirm the pathogenicity, Bavendamm assay was conducted for measuring phenoloxidase activity. The colony size of the CpSlp1-null mutant was not different from that of EP155/2, but the CpSlp1-null mutant’s level of brown coloration was higher than EP155/2. And the directly virulence assay conducted on the chestnut tree bark revealed that the CpSlp1-null mutants produced similar size of necrotic areas compared with the wild type EP155/2. Thus, our study suggests that the protein product of CpSlp1 is involved in growth and cell wall composition, has a role in phenoloxidase activity but does not affect pathogenicity. 밤나무 줄기 마름병을 일으키는 사상성 진균인 Cyphonectria parasitica와 hypovirus인 Cryphonectria Hypovirus 1 (CHV1)은 곰팡이-바이러스 상호작용 메커니즘을 연구하는 데 유용한 모델이다. 이전 실험실 연구에서, cell wall integrity pathway와 관련된 두 개의 유전자(CpBck1, CpSlt2) 돌연변이에서 이상분화(sectorization)가 관찰되었다. 이상분화의 특징으로는 야생형(EP155/2)에 비해 병원성이 감소한 반면 성장(growth)은 회복되었으나 발달(development)이 일어나지 않는 특징을 보였다. 또 다른 선행연구에서, 이상분화가 나타난 곳에서 분리된 자손과 해당 부모 돌연변이 균주사이에서 73개의 공통적인 차등 발현 유전자(DEG)를 RNA 시퀀싱 분석을 통해 밝혀냈고 이 73개의 차등 발현 유전자중 22개의 유전자가 CHV1 감염에 영향을 받는 것으로 나타났다. 본 연구에서는 이상분화와 CHV1 둘 다에 영향을 받는 22개의 유전자중 하나인 Stomatin-like protein (CpSlp1)을 선정하여 실험을 진행하였다. BLAST 검색을 통해 유전자가 Coniella lustricola의 membrane protein family와 가장 높은 유사성을 보였다. Cryphonectria parasitica의 Stomatin-like protein의 기능을 분석하기 위해, 상동 재조합을 통한 유전자 제거를 하여 192개의 후보군중 1개의 CpSlp1-null 돌연변이를 얻었다. CpSlp1-null 돌연변이는 PDAmb에서 EP155/2와 형태가 유사했지만, 성장률은 더 적었다. 또한 세포벽 형성을 방해하는 시약이 첨가된 배지에서도 역시 성장률이 더 적었다. 하지만, 삼투압 스트레스나 활성 산소(ROS)에 대한 스트레스 반응에서는 유의미한 차이가 나타나지 않았다. 병원성을 알아보기 위한 실험에서는 간접적으로 병원성을 측정하는 Bavendamm assay를 통해CpSlp1-null 돌연변이가 EP155/2와 비교했을 때 더 진한 갈색을 나타나는 것을 알 수 있었지만, 직접적인 밤나무 접종 실험에서는 Bavendamm assay의 결과와 다르게 유의미한 차이가 나타나지 않았다. 따라서, 이 연구는 CpSlp1 유전자가 성장과 세포벽 구성에 관여하고, 페놀옥시다아제(phenoloxidase) 활성에 중요한 역할을 하지만 실제로는 병원성에는 직접적인 영향을 미치지 않는다는 것을 시사한다.

ROLE OF INTERLEUKIN (IL)-1α AS A LOCAL REGULATOR OF PROSTAGLANDIN AND PLASMINOGEN ACTIVATOR IN BOVINE ENDOMETRIUM

타니카와 미치요 강원대학교 대학원 2008 국내박사

These studies were performed to understand the role of interkeukin (IL)-1a as a local regulator of prostaglandin and plasminogen activator in bovine endometrium. In chapter 1, the effects of IL-1a and IL-1b on PGF2a and PGE2 production and plasminogen activator (PA) activity in cultured bovine endometrial epithelial and stromal cells as well as the expression of IL-1a, IL-1b and IL-1 receptor type 1 (IL-1R) mRNA in cultured bovine endometrial cells by RT-PCR were examined. RT-PCR revealed that mRNA of IL-1a only in the stromal cells, and that IL-1b and IL-1R mRNA is expressed in both type of cells. Cultured bovine epithelial and stromal cells were exposed to IL-1a and IL-1b (0.006-3 nM) for 24 h. IL-1a and IL-1b dose-dependently stimulated PGE2 and PGF2a production in the stromal cells (P<0.05), whereas the stimulatory effect of IL-1a and IL-1b on both PG productions were not observed in epithelial cells. On the other hand, IL-1a and IL-1b dose-dependently increased PA activity in the epithelial cells, whereas the stimulatory effect of both IL-1 on PA production was not observed in stromal cells. When the stromal cells were exposed to 0.06-3 nM IL-1a and IL-1b for 24h, there was a significant difference in the both PG production between IL-1a and IL-1b treated cells. In epithelial cells, there was a significant difference in PA production between IL-1a and IL-1b treated cells when the cells were exposed to 0.6-3 nM IL-1a and IL-1b for 24h. The overall results suggest the possibility that both IL-1 are produced by the epithelial (only IL-1b) and stromal cells, and IL-1a is far more potent stimulator than IL-1b on PG and PA production in cultured bovine endometrial epithelial and stromal cells. Chapter 2 was performed to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGE2 and PGF2a in response to IL-1a, and the intracellular mechanisms of IL-1a action. Cultured bovine epithelial and stromal cells were exposed to IL-1a (0.006-3.0 nM) for 24 h. IL-1a dose-dependently stimulated PGE2 and PGF2a production in the stromal cells (P < 0.05), but not in the epithelial cells. The stimulatory action of IL-1a on PG production was augmented by supplementing arachidonic acid (AA). When the stromal cells were incubated for 24 h with IL-1a and inhibitors of phospolipase (PL) C or PLA2 (anthranilic acid; 1 uM), only PLA2 inhibitor completely stopped the stimulatory action of IL-1a on PG production. Moreover, a specific cyclooxygenase (COX)-2 inhibitor blocked the stimulatory effect of IL-1a on PG production. IL-1a (0.06-0.6 nM) dose-dependently promoted COX-2 and microsomal PG synthase-1 (mPGES-1) gene expressions. The expression of PGFS mRNA was not affected by IL-1a (0.06-0.6 nM) in the stromal cells. IL-1a (0.06-0.6 nM) increased COX-2 protein expression in the stromal cells (P<0.05). mPGES-1 protein expression was significantly increased by 0.06 nM IL-1a (P<0.05) but not 0.6 nM IL-1a. PGFS protein expression was significantly stimulated by 0.6 nM IL-1a (P<0.05). The overall results indicate that 1) the target of IL-1a for stimulating both PGE2 and PGF2a production is the stromal cells, 2) the stimulatory effect of IL-1a on PG productions is mediated via the activation of PLA2 and COX-2, 3) IL-1a induced PG production by increasing expressions of COX-2 mRNA and its protein, and by differently modulating mRNA and protein expressions of mPGES-1 and PGFS in bovine stromal cells. Chpter 3 was conducted to determine the signal transduction pathways involved in PGE2 production via the activation of COX-2 and mPGES-1 by IL-1a in bovine endometrial stromal cells. Specific inhibitors for ERK1/2 (U0126; 10 uM) and p38 MAPK (SB203580; 10 uM) significantly inhibited both basal and IL-1a-induced PGE2 production in bovine stromal cells (P< 0.05). However, specific inhibitor for PI3 kinase (LY294002; 10 uM) stimulated both basal and IL-1a-induced PGE2 production in bovine stromal cells. U0126 and SB203580 significantly inhibited both basal and IL-1a-induced COX-2 mRNA and protein expressions in bovine stromal cells (P< 0.05). However, basal and IL-1a-induced mPGES-1 mRNA and protein expressions were reduced by only U-0126, but not by an inhibitor for SB203580 in bovine stromal cells. LY294002 enhanced both basal and IL-1a-induced COX-2 and mPGES-1 mRNA and protein expressions in bovine stromal cells. The overall results indicate that 1) IL-1a-induced COX-2 expression is via activation of both ERK1/2 and p38 MAPK, 2) IL-1a-induced mPGES-1 expression is via activation of only ERK1/2 in stromal cells. In other word, IL-1a may use overlapping, but distinct signaling, pathway to induce COX-2 and mPGES-1 expressions in bovine endometrial stromal cells. In Chapter 4, the effect of IL-1a on PA activity, especially focusing on the effect of IL-1a on mRNA and protein expression of key factors directly involved in PA activity such as t-PA, u-PA, PAI-1 and PAI-2 were determined. The signal transduction pathway involved in IL-1a action on PA activity via phosphorylation of signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 (PI3) kinase were also studied. Cultured bovine epithelial and stromal cells were exposed to IL-1a (0.006-3.0 nM) for 24 h. IL-1a dose-dependently stimulated PA activity in the epithelial cells (P < 0.05), but not in stromal cells. IL-1a (0.6 nM) promoted t-PA, u-PA and PAI-2 gene expressions. The expression of PAI-1 was not affected by IL-1a. Specific inhibitors for ERK1/2 (U0126; 20 uM) and p38 MAPK (SB203580; 20 uM) significantly inhibited both basal and IL-1a-induced PA activity in bovine epithelial cells (P< 0.001). However, specific inhibitor for PI3 kinase (LY294002; 20 uM) did not affect both basal and IL-1a-induced PA activity in bovine epithelial cells.

Correlation between age-related inflammation and visceral lipid accumulation via SREBP-1 and PPARα/β

김지영 부산대학교 대학원 2007 국내박사

Accumulated evidence indicates that the pattern of lipid accumulation undergoes substantial changes during aging, which may play a major role in the age-related dyslipidemia and metabolic syndrome, as seen in the adult-onset diabetes and obesity. Evidence further reveals that inflammation of adipose tissues, particularly visceral tissue, may act as a potent mediator is associated with this age-related altered lipid accumulation, i.e. redistribution. However, the molecular information on the interplay between this age-related lipid redistribution and inflammation is lacking at the present. Animals utilized for the current study were Fischer 344 male rats fed ad libitum (AL) or calorie restriction (CR, 40% less than AL) at 5-, 17-, and 24-month-old of age, and ob/ob mice. In the first phase of the present study, changes in age-related lipid accumulation pattern was explored by focusing on key mediators, sterol regulatory element binding protein-1 (SREBP-1), peroxisome proliferators-activated receptor alpha/beta (PPARalpha/beta) and adipose triglyceride lipase (ATGL) that are known to play major roles in the lipid distribution. Results from the first phase on lipid distribution revealed that the adipose lipid accumulation was reduced by the decreased levels of SREBP-1, PPAR gamma2 activities and ATGL during aging. To monitor the lipid accumulation in non-adipose tissues, the fatty acid uptake by skeletal muscle and heart was assessed, and found the uptake increased with age. However, PPAR alpha/beta and carnitine palmitoyltransferases-1 significantly decreased in skeletal muscle and heart, while levels of SREBP-1 and glycerol-3-phosphate acyltransferase and triglyceride (TG) levels increased in aged skeletal muscle and heart. These data from the first phase of the study strongly suggest that fatty acids that were not taken up by adipose tissues were stored into skeletal muscle and heart, thus showing the molecular insights on the altered lipid accumulation pattern during aging. For the question on the visceral lipid accumulation and inflammation, attention was given to several key proinflammatory factors such as nuclear factor-kappaB, tumor necrosis factor-alpha, cyclooxygenase-2, and monocyte chemoattractant protein-1, and found that they were all augmented with age in skeletal muscle and heart. To explore the correlation between visceral lipid accumulation and inflammation, proinflammatory factors were examined using ob/ob mice that have abundant visceral adiposity. Results showed that proinflammatory factors and F4/80, marker for macrophage in mouse, significantly augmented in ob/ob mice. It was also found that in skeletal muscle and heart of ob/ob mice, parameters related to fatty acid uptake and TG levels increased. Most significant finding of the present study is the modifying action of CR that is known to exert against the diabetics, obesity and inflammation on the lipid distribution and adipose tissue-related inflammation. Under CR, it was found that the altered lipid accumulation pattern was effectively modulated, which is closely related to the CR’s attenuation of inflammation. Furthermore, data on SREBP-1 and PPAR alpha/beta indicate that the interplay between visceral lipid accumulation and inflammation is linked through the induction of SREBP-1 and PPAR alpha/beta. Significance of the current findings is the relation between visceral lipid accumulation and age-related inflammation, thus providing a better molecular understanding of the lipid distribution during the aging process and possibly age-related obesity.

(The) p150 large subunit of chromatin assembly factor 1 regulate ATM pathway in double strand breaks repair.

조병옥 조선대학교 대학원 2008 국내박사

Chromatin assembly factor 1 (CAF-1)은 게놈 보전과 DNA 수복에 관여하는 중요한 유전자다. 그렇지만, CAF-l이 DNA 수복에 어떻게 관여하는지는 많이 연구되어지지 않았다. 본 연구에서는 DNA double strand breaks (DSBs) 수복에서 CAF-1 p150 의 생물학적인 역할을 연구했다. CAF-1 p150은 DSBs 수복 반응에서 우선적으로 활성화 되어지는 ATM (ataxia telangiectasia mutated) 유전자와 직접 상호작용을 한다. 또한, CAF-1 p150은 DSBs 수복이 일어나는 곳에 보충되고, 동시에 ATM과 함께 집중된다. RNA interference를 통한 CAF-1 p150의 silencing은 ATM과 H2AX의 인산화를 감소시켰고, foci 형성 또한 약간 감소시켰다. 그러나, 53BP1, MDC1, BRCA1, Chk2 and MRN complex같은 DSBs 수복에 관여하는 DNA 수복 단백질의 보충에는 영향을 미치지 않았다. 또한, CAF-1 p150이 knockdown된 세포에서 Chk2의 인산화는 지연되었고 감소되었다. 그렇지만, BRCA1과 p53의 인산화는 증가되었다. DSBs를 일으키는 물질인 Neocasinostatin (NCS)를 처치한 후에 DNA가 손상된 세포는 G2/M기에서 세포주기가 정지하였다. 이러한 결과는 DSBs 수복에서 CAF-1 p150이 ATM 신호전달 경로를 조절함으로써 세포주기 정지에 관여한다는 것을 나타낸다. 결론적으로, DNA double strand breaks 수복에서 CAF-1 p150은 G2/M기 DNA checkpoint를 조절하고 ATM 신호전달 경로를 통해서 DNA 수복에 관여할 것으로 사료된다. Chromatin assembly factor 1 (CAF-1) is involved in genome integrity and DNA repair. Here we investigated the biological role of CAF-1 p150 to DNA double strand breaks (DSBs) in HeLa cell. CAF-1 p150 directly interacted with ATM ( ataxia telangiectasia mutated), which is primarily activated in response to DSBs. CAF-1 p150 is also recruited to foci with kinetics to, and colocalizes with ATM in response to DSBs. Silencing of CAF-1 p150 via RNA interference decreased phosphorylation levels of ATM and H2AX and foci formation a little, but didn’t affect to recruit another DNA repair protein including 53BP1, MDC1, BRCA1, Chk2 and MRN complex in response to DSBs, although BRCA1 foci slightly induced. Chk2 phosphorylation were delayed and decreased in CAF-1 p150 depleted cells. However, BRCA1 and p53 phosphorylation surprisingly increased in CAF-1 p150 depleted cells. DNA damaged cells after necocasinostatin (NCS) treatment causing DSBs arrested G2/M phase in cell cycle. These results indicate that CAF1 p150 involved in cell cycle arrest by regulating ATM signaling pathway in DSBs. Collectively, our results suggest that CAF-1 p150 regulates G2/M phase DNA checkpoint and involved in DNA repair through ATM signaling pathway in DNA double strand breaks.

Protective Roles and Regulation of Rad9 and Spy1 from the fission yeast

강민희 강원대학교 대학원 2008 국내석사

PART I. Protective Role and Regulation of Rad9 from the fission yeast Schizosaccharomyces pombe. 세포 주기내의 checkpoint protein으로 알려져 있는 Rad9은 DNA 손상에 반응 하여 인산화 된다. 분열 효모인 Schizosaccharomyces pombe 내의 rad9 유전자의 특성과 조절에 대해 조사하기 위하여 분열 효모의 genomic DNA로부터 구조 유전자를 증폭하였고, 이를 shuttle vector인 pRS316과 ligation을 통해 재조합 된 plasmid pYFRad9을 생성하였다. Cloned rad9 gene은 2,252 bp의 nucleotide sequence를 포함하는 데, 426개의 아미노산을 갖는 단백질을 encoding하게 되며, 이 단백질의 분자량은 47,494 Da이다. 또한, 이 DNA 서열에는 651 bp의 upstream 부분과 153 bp의 downstream 부분을 있으며, 3개의 intron이 존재한다. Schizosaccharomyces pombe 내에 재조합 pYFRad9을 도입한 후 Rad9의 mRNA 발현수준 정도를 조사 한 결과 pYFRad9의 mRNA 발현이 증가하여, 세포내의 여러 생리적 기능을 담당할 것이라는 사실을 알 수 있다. rad9 유전자의 생리적 기능을 확인 하기 위해서 다양한 스트레스를 주어 S. pombe cell의 viability에 영향을 주는지 cell survival test를 통하여 확인하였다. nitric oxide (NO)-generating sodium nitroprusside (SNP), hydrogen peroxide (H2O2), cadmium chloride(CdCl2)와 같은 다양한 스트레스 유발 물질로부터 rad9 유전자가 도입된 cell이 control vector인 pRS316만 도입된 cell에서보다 증가된 cell survival을 나타내고 있음을 확인하였고, 이 중 10 μM 과 20 μM SNP 조건에서의 각각 35 %와 60 % viability를 나타냄을 확인하였다. 세포가 성장과 번식하는데 필요한 nutritional source의 하나인 nitrogen을 결핍 시킨 조건 하에서의 viability 또한 rad9 유전자가 도입된 cell이 control vector만 도입된 cell에서보다 또한 높은 viability를 가짐을 확인하였다. 이러한 사실들로부터 S. pombe rad9 유전자가 여러 스트레스로부터 세포를 보호하는데 있어서 중요한 역할을 할 것이라는 사실을 알 수 있다. 651 bp upstream region은 fusion plasmid를 만들기 위하여 shuttle vector YEp367R의 promoter가 없는 β-galactosidase 유전자와 fusion하였다. Fusion plasmid pYURad9를 Pap1-positive cells과 Pap1-negative cells에 transformation을 진행하였다. Fusion plasmid pYURad9성질 전환된 Pap1-positive cell으로부터 합성되는 β-galactosidase의 합성은 높은 viability를 보였던 SNP와 nitrogen starvation 조건에서 증가하였다. 그러나 fusion plasmid pYURad9성질 전환된 Pap1-negative cells에서의 β-galactosidase의 합성은 증가되지 않았다. 이를 RT-PCR로 통해서 mRNA level을 확인해 본 결과 그 발현을 증가 시키는 것을 확인 하였다. 또한 낮은 농도의 glucose와 sucrose (0.2 %) 조건하에서는 Pap1-positive cell에서의 β-galactosidase의 합성이 증가 되어짐을 확인하였고, Pap1-negative cells에서의 합성은 증가되지 않았다. 하지만, 다른 agent인 menadione(Md), mercuric chloride(HgCl2), hydrogen peroxide(H2O2)에 의해서는 Pap1-positive cell과 Pap1-negative cells 모두에서 β-galactosidase의 합성은 유도되어지지 않았음을 확인하였다. Glutathione (γ-glutamyl-L-cysteinylglycine; GSH)의 수준을 낮춤으로써 핵 내의 전사조절인자인 Pap1의 축적을 유도 한다고 알려진 diethylmaleate(DEM)을 Pap1-positive cell과 Pap1-negative cells에 처리 함으로써 Pap1에 dependent한 manner에서 Rad9은 stress 반응에 대하여 방어 기작을 가지는 것을 재 확인 하였다. 하지만 이들은 ROS의 생성을 낮추는 역할을 하지 않은 것으로 확인 하였다. PART II. Physiological Roles and Regulation of Spy1 in Schizosaccharomyces pombe. 분열효모 Schizosaccharomyces pombe 내의 스트레스 반응 조절 유전자로 추정 되는 Spy1이라는 새로운 유전자의 기능을 규명하기 위해 실험을 진행하였다. 분열 효모의 genomic DNA로부터 구조유전자를 증폭하였고, 이를 shuttle vector인 pRS316과 ligation을 통해 재조합 된 plasmid pYFSpy1을 생성하였다. Cloned Spy1 gene을 1,437 bp의 nucleotide sequence를 포함한다. 이 중 295 아미노산 서열부분이 암호화되었으며, 이 분자의 분자량은 32,550 Da이다. 아미노산 서열은 phosphotransmitter protein인 YPD1으로 추정 되어지는 Aspergillus clavat NRRL1과 Saccharomyces cerevisiea YPD1와 높은 homology를 가진다. 또한, 이 DNA 서열은 1,233 bp의 upstream 부분을 가지고 있다. Schizosaccharomyces pombe 내에 재조합 된 pYFSpy1을 도입한 후 Spy1의 mRNA 발현수준 정도를 조사 한 결과 pYFSpy1의 mRNA 발현이 증가하였다. 생리적 기능을 확인 하기 위해서 cell survival test를 통하여 확인하였다. nitric oxide (NO)-generating sodium nitroprusside (SNP), hydrogen peroxide (H2O2), cadmium chloride(CdCl2)를 처리하여 다양한 스트레스 유발 하였을 때, Spy1 유전자가 도입된 cell이 control vector인 pRS316만 도입된 cell에서보다 증가된 cell survival을 나타냄을 확인 하였다. 20 μM SNP와 nitrogen starvation 상황에서의 viability역시 증가함을 통해 Spy1이 스트레스에 대하여 조절 기작을 가지는 것으로 확인 하였다. 유전자의 특징, 조절 양상을 규명하고자 분열효모 Spy1 유전자의 1,233 bp upstream region만을 증폭하여 YEp367R shuttle vector 와 ligation을 통해 재조합 된 pYUSpy1을 생성하였다. S. pombe Spy1 유전자의 다양한 stress에 의한 발현 조절을 독립적으로 확인하기 위하여 LacZ-Spy1 fusion plasmid를 조성하였다. 이렇게 조성된 fusion plasmid pYUSpy1을 분열효모 Pap1-positive KP1(h+ leu-32 ura4-294)과 Pap1-negative TP108-3C(h- leu1 ura4 pap1::ura4+)에 도입하여 Spy1 유전자의 발현을 검토하였다. SNP와 nitrogen starvation 조건에서 Pap1-positive KP1 cells에서의 β-galactosidase의 합성은 유도가 증가됨을 확인 하였다. Menadione(Md)으로부터 유도 되어지는 ROS의 생성을 Spy1이 유도 되어진 cells에서 ROS의 level을 감소 시키는 기능을 확인하였다. 또한, cell survival의 효과가 있었던 25 μM cadmium chloride(CdCl2)을 처리하였을 때 mRNA의level이 증가함을 RT-PCR을 통하여 확인하였다. SNP로부터 방출되는 nitric oxide (NO)의 생성을 scavenger 한다는 것을 확인 함으로써 S. pombe에 존재하는 Spy1의 세포 내 존재 이유와 cell내에서의 각기 다른 기능과 조절의 차이를 연구하는데 중요한 정보를 기대할 것으로 기대된다. PART I. Protective Role and Regulation of Rad9 from the fission yeast Schizosaccharomyces pombe. To assess novel cellular roles and regulation of Rad9 in the fission yeast Schizosaccharomyces pombe, the full-length rad9 gene was cloned into the shuttle vector pRS316, generating pYFRad9. The rad9 mRNA level was significantly increased in the S. pombe cells harboring the plasmid pYFRad9, suggesting that the cloned rad9 gene is functioning. The S. pombe cells harboring pYFRad9 showed higher survival in the minimal media containing NO-generating sodium nitroprusside (SNP, 20 μM) and no nitrogen than the vector control cells. SNP and nitrogen starvation notably enhanced the synthesis of β-galactosidase from the rad9-lacZ fusion gene in the Pap1-positive cells but not in the Pap1-negative cells. The rad9 mRNA level, detected by semi-quantitative RT-PCR, was elevated in the Pap1-positive cells but not in the Pap1-negative cells by SNP and nitrogen starvation. It was also increased only in the Pap1-positive cells by diethylmaleate which activates Pap1. Collectively, the results imply that Rad9 plays a protective role against nitrosative and nutritional stress and is positively regulated by nitric oxide and nitrogen starvation in a Pap1-dependent manner. PART II. Physiological Roles and Regulation of Spy1 in Schizosaccharomyces pombe. This work was designed to assess physiological roles and regulation of Spy1, a histidine-containing phosphotransfer (HPt) protein, in the fission yeast Schizosaccharomyces pombe. The structural gene encoding Spy1 was transferred into the shuttle vector pRS316 to generate the recombinant plasmid pYFSpy1. The Spy1 mRNA level was notably increased in S. pombe cells harboring the plasmid pYFSpy1, suggesting that the cloned Spy1 gene is functioning. The S. pombe cells harboring pYFSpy1 showed higher survival than the control cells on the minimal media plates containing hydrogen peroxide, cadmium or nitric oxide-generating sodium nitroprusside (SNP). They also showed higher viability under nitrosative stress or nitrogen-starved condition. The reactive oxygen species (ROS) level was lower in the fission yeast cells harboring pYFSpy1 than in the control yeast under normal and even stressful conditions, such as menadione and nitrogen starvation. The synthesis of β-galactosidase from the Spy1-lacZ fusion gene was significantly enhanced by SNP and nitrogen depletion. It was also shown that the Spy1 mRNA level in S. pombe was elevated by cadmium. Spy1 showed scavenging effect on nitric oxide (NO) generated from SNP. Taken together, Spy1 plays a protective role against oxidative, nitrosative and nutritional stress in the fission yeast and its gene is transcriptionally regulated by stress in a positive manner.

Astrocytes의 hemeoxygenase-1의 발현을 통한 microglia의 활성화조절

민경진 아주대학교 2007 국내박사

In damaged brain, inflammation occurs as a host defense mechanism. However, brain inflammation has a double-edged effect on brain damage. Inflammation protects the brain from infection, but aggravates the injury of surrounding tissue. Therefore, negative regulatory mechanisms of brain inflammation exist to prevent prolonged and extensive inflammation. The first part of this thesis showed that astrocytes, the most abundant cells in the brain modulate microglial activation by regulating the microglial levels of reactive oxygen species (ROS). Astrocyte culture-conditioned media (ACM) suppressed IFN-gamma- nor LPS-induced ROS production, leading to reduced iNOS expression and NO release in microglia. Treatment of microglia with ACM increased the expression level and activity of hemeoxygenase-1 (HO-1) through the activation of nuclear factor E2-related factor 2 (Nrf2) transcription factor. The active component(s) in ACM was/were heat-labile and smaller than 3 kD. Furthermore, phosphoinositide-3 kinase (PI3K) mediates ACM-induced HO-1 expression. Mimickers of HO-1 products such as bilirubin, ferrous iron and a carbon monoxide (CO)-releasing molecule also reduced IFN-gamma-induced iNOS expression and/or NO release. Damaged astrocytes and neurons also down-regulate microglial activation through HO-1 expression. Therefore, astrocytes and neuron appears to cooperate with microglia to prevent excessive inflammatory responses in the brain by regulating microglial ROS production. The second part of this thesis showed that adenosine could modulate microglial activation. In response to adenosine, Nrf2 was translocated from the cytosol to the nuclei, bound to ARE, and then HO-1 promoter activity increased. PI3K and its downstream, Akt, appeared to mediate adenosine-induced HO-1 expression since adenosine induced Akt phosphorylation and inhibitors of PI3K (LY294002 and wortmanin) reduced adenosine-induced HO-1 expression. These results suggest that adenosine could be an endogenous regulator of brain inflammation through the modulation of microglial ROS production. Taken together, these results suggest that astrocytes and neuron, and an endogenous factor, adenosine, could regualte microglia-mediated brain inflammation to minimize inflammation-induced brain damage.

Characterization of Pichia pastoris PGK1 promoter and its application for heterologous protein expression

Lee, Sung-Jae 강원대학교 대학원 2007 국내석사

3-Phosphoglycerate kinase (PGK)는 해당과정에 한 역할을 담당하는 효소로, yeast 전체 세포내 단백질의 5%정도 존재 한다. PGK는 세포내에서 발현양이 많기 때문에 이 효소의 promoter를 이용한 외부단백질 발현 vector는 많은 연구가 되어져 왔다. 그러나 Pichia pastoris의 PKG1 promoter는 흔히 사용되는 제한효소 부위가 많았고, 또 2.0 kb나 되는 sequence를 실제 vector에 사용하는 데는 무리가 있었다. 본 연구는 2.0 kb의 Pichia pastotis의 PGK1 promoter를 활용하기 위하여 5‘부터 단계적으로 제거하고자 함으로 발현에 영향을 주는 부위를 분석하였다. 이를 위해 시작 코돈으로부터 -0.25와 -0.5, 0.75, 1kb 부위에 primer를 제작하였고, PCR 방법을 통해 여러 크기의 PGK1 promoter를 얻었다. 이 PGK1 promoter들을 pGAPZB의 GAP promoter와 교체하였고, Pichia specific autonomous replication sequence (PARS1)를 삽입하여 PGK1 promoter episomal vector를 만들었다. 이 vector에 외래 단백질 lacZ와 GFPuv를 삽입하여 분석하였다. 분석 결과 PGK1 promoter를 0.25kb 크기로 줄였을 때에도 2.0kb의 promoter와 protein 활성이 다르지 않음을 확인하였다. 또 0.25kb PGK1 promoter내에 -174 bp XbaI 제한효소 부위의 한 서열을 변형시켜본 결과 단백질활성이 떨어짐을 확인하였다. 이를 통해 PGK1 promoter의 size를 025 kb까지 줄였고, 이는 0.75 kb의 Saccharomyces cerevisiae와 비교해볼 때 작은 크기다. 이렇게 제조된 0.25kb의 PGK1 promoter vector에 인간 단백질 HR969을 적용하여 보았다. SDS-PAGE 분석 결과 17 kDa의 HR969로의 단백질밴드를 확인 할 수 있었다. 또 PGK1 promoter를 Saccharomyces cerevisiae의 secretion sequence인 α-factor를 이용하여 HSA를 발현에 적용하여 보였고, 66,5 kDa의 HSA 단백질 밴드를 확인하였다. PGK1 promoter를 다른 constitutive promoter인 GAP promoter와 비교해 보았다. β-galactosidase나 GFPuv를 발현하였을 때, GAP promoter를 이용시 발현양이 많았고, HR969 protein의 경우에는 비슷하였다. 그러나 외부단백질 발현에서 GAP promoter를 이용할 경우는 cell이 하루 정도 늦게 자라는 반면 PGK1 promoter는 세포성장에 큰 영향을 주지 않았다. HSA의 외래 발현에서는 PGK1 promoter를 이용할 때가 GAP promoter를 이용할 때 보다 더 많은 단백질 발현양을 보였다.

습식법에 의한 고효율 망가니즈산화물 흡착제의 합성 및 물성연구

이환 건양대학교 대학원 2007 국내석사

High-efficiency manganese oxide adsorbents for the selective recovery of lithium ion in seawater was prepared and its lithium adsorption properties were examined. Li1.6Mn1.6O4 precursor was synthesized by the wet process. The adsorbents, H1.6Mn1.6O4 was derived from Li1.6Mn1.6O4 by acid treatment. The spinel structure of precursor was obtained by heat treatment at 500℃ for 4hrs. An optimum ion sieve was obtained by 3 days of 0.5 M-HCl treatment with stirring. It had good selectivity and high efficiency in adsorbing lithium ion in seawater. The generated adsorbent showed a 44.3 mg/g lithium uptake from artificial seawater. 해수 중에 포함되어 있는 리튬에 대한 선택적인 회수를 위하여 고효율 망가니즈산화물 흡착제를 제조하고 리튬 이온에 대한 흡착특성을 연구하였다. 습식법에 의하여 Li1.6Mn1.6O4 전구체를 합성한 뒤 산 처리를 통하여 H1.6Mn1.6O4 흡착제를 제조하였다. 전구체의 스피넬 결정형성의 최적의 조건은 500 ℃에서 4시간 동안 열처리하여 얻을 수 있었으며, 최적의 이온체 형성은 0.5 M염산용액에서 리튬 이온을 1 회당 24 시간씩 3 회 교반, 침출시켜 냄으로써 얻었다. 제조된 H1.6Mn1.6O4 흡착제는 해수에서 리튬 이온에 대한 탁월한 선택성과 높은 효율을 나타내었으며 인공해수로부터 단위 그램당 44.3 mg의 리튬 이온을 흡착시킬 수 있는 우수한 흡착능을 나타내었다.

고양이의 치수에서 capsaicin 유도성 혈관확장에 대한 TRPV1 수용체의 영향

우준하 경북대학교 일반대학원 2007 국내석사

본 연구의 목적은 capsaicin에 의해 유도된 혈류량의 변화에 TRPV1 수용체가 관여하는지를 연구함으로서 고양이의 치수에서 TRPV1 수용체의 기능적 역할을 탐구하고자 하였다. 12마리의 고양이가 전신 마취하에 사용되었다. 전신 혈압과 치수 혈류량을 관찰하기 위해 악골을 고정하였다. 치과용 버를 이용하여 하악 견치의 상아질을 노출시키고 laser Doppler flowmetry (Periflux 4001, Perimed., Stockholm, Sweden)로 치수 혈류량의 변화를 관찰하였다. 치수 혈류량에 대한 capsaicin의 영향을 평가하기 위해 capsaicin (0.2-0.8 μg/kg)을 40초 동안 일정한 속도로 상악동맥을 통해 주입하였다. 대조군으로 생리식염수가 사용되었다. 치수 혈류량의 변화에 TRPV1 수용체가 관여함을 평가하기 위해 TRPV1 길항제인 5-iodoresiniferatoxin (I-RTX, 1 μg/kg)를 상악동맥을 통해 주입하고 10분 후에 capsaicin (0.2-0.8 μg/kg)을 주입하였다. 약물을 투여하기 전에 안정적인 laser Doppler 신호를 대조치로 하고, 약물 투여 직후 최대 변화값을 실험치로 하였다. 결과는 one-way ANOVA와 paired t-test로 분석하여 다음과 같은 결과를 얻었다. Capsaicin (0.2-0.8 μg/kg)의 투여는 30 ± 3.2 %의 치수 혈류량 상승을 초래하였고 (p<0.001), 6.3 ± 2.1초 동안 지속되었다. 생리식염수와 I-RTX 자체는 치수 혈류량의 유의한 변화를 초래하지 않았다(p<0.001). 상악동맥을 통해 I-RTX를 주입 후 capsaicin을 투여한 경우에는 치수 혈류가 9.8 ± 1.5 % 만 증가하였다(p<0.001). 따라서 TRPV1 수용체의 길항제인 I-RTX는 capsaicin으로 인한 치수 혈류량의 증가를 현저히 억제하였다. 따라서 본 연구의 결과로 보아 고양이에서 capsaicin 유도성 TRPV1 수용체가 치수 혈류변화 조절에 기능적으로 관여함을 알 수 있었다.

세포 실험을 이용한 돼지감자 추출물의 항비만/당뇨 효과 검증

김정란 전북대학교 교육대학원 2008 국내석사

This study was designed to evaluate the anti-obesity and anti-diabetes effect of Helianthus tuberosus extract(HT) in 3T3-L1 cells and HIT-T15 cells. The experimental groups were divided into 9 groups; NC(0 ㎕/㎖), HT2(1.1 ㎕/㎖), HT3(1.5 ㎕/㎖), HT4(2 ㎕/㎖), HT5(2.4 ㎕/㎖), IN2(1.8 ㎕/㎖), IN3(2.5 ㎕/㎖), IN4(3.2 ㎕/㎖), IN5(4 ㎕/㎖). Inulin(IN) was used as a positive control of Helianthus tuberosus extract groups. Measured of oil red O stain, triglyceride(TG), leptin level and gene expressions on peroxisome proliferator-activated Receptor-γ(PPAR?) acetyl CoA carboxylase(ACC), Carnitine palmitoyltransferase-I(CPT-1) in 3T3-L1 cells. Confirmed of cell viability, cytotoxicity in HIT-T15 cells and cell survival, insulin secretion level, NAD+/NADH ratio in HIT-T15 cells treated alloxan. There was no significant difference in oil red O stain in all groups. Triglyceride(TG) was significantly decreased in the IN2, IN3 IN4 groups, compared with the NC group. Leptin level was significantly decreased in the IN2, IN3 groups, compared with the NC group. The mRNA expression of PPAR? and ACC were increased in the HT5 group, compared with the NC group. CPT-1 mRNA expression was higher in the HT3 group than the NC group. Cell viability was significantly increased in the HT2, HT3, HT4, IN2, IN3 groups, compared with the NC group. There was no significant difference in cytotoxicity in all groups. Cell survival and NAD+/NADH ratio were significantly increased in the HT3, HT4, IN2, IN3 groups, compared with the NC group. Insulin secretion was significantly increased in the HT2, HT3, HT4, IN2 groups, compared with the NC group. In conclusion, the Helianthus tuberosus extract supplementation was ineffective in the cell differentiation of 3T3-L1 cells. Whereas, We have demonstrated that Helianthus tuberosus extract supplementations of low concentration in HIT-T15 cells have beneficial effects. It provide that the Helianthus tuberosus extract supplementations of low concentration in HIT-T15 cells increased cell viability, protective effect of β-cell, insulin secretion level and NAD+/NADH ratio. Finally, these results may suggest that Helianthus tuberosus extract supplementations of low concentration possesses anti-diabetes effects and control blood sugar.