Functional characterization of virulence factors, thermostable direct hemolysin (Tdh) and calcium response protein D1 (VcrD1) in vibrio parahaemolyticus

Dept. of Medical Science/석사Vibrio parahaemolyticus, a gram-negative marine bacterium, is a worldwide cause of foodborne gastroenteritis. Thermostable direct hemolysin (Tdh) is a factor responsible for the Kanagawa phenomenon caused by pathogenic V. parahaemolyticus.In chapter I, expression of Tdh was examined under various culture conditions. Two tdh genes, tdhA and tdhS, were identified in the genomic sequence of wild-type V. parahaemolyticus. Reporter fusions between these tdh promotors and the luciferase gene indicated that the tdhA gene is expressed higher than tdhS under the conditions tested, and expression of both tdh genes was induced by the presence of an iron chelator or crude bile in the medium. Candidate transcription factor(s) for bile-mediated tdh expression were isolated by ligand fishing experiments. Through Western blot analysis and fusion assay on toxR mutant V. parahaemolyticus, a global regulator, ToxR (cholera toxin transcriptional activator) was found to control bile-induced expression of the tdhA gene. Direct binding of ToxR protein to the tdhA promoter was shown by gel-shift assays. In addition, the cytotoxic role of TdhA was observed by treating human gastrointestinal HT-29 cells with recombinant TdhA (rTdhA). HT-29 cells treated with rTdhA demonstrated considerable morphological changes under transmission electron microscopy. Both apoptotic and necrotic cell deaths were detected by staining with Annexin V and PI, respectively. Cytotoxicity of rTdhA was reduced by the RIP-1-mediated necroptosis inhibitor, necrostatin-1, indicating that rTdhA causes host cell death by necroptosis. These data clearly showed that expression of Tdh is modulated by certain physiological signals, such as iron depletion and presence of bile, and this toxin is quite capable of killing host cells mainly via necrosis.In chapter II, VcrD1 protein is highlighted as a component of type III secretion system (T3SS) 1 in Vibrio parahaemolyticus. A comparative analysis of secretomes of wild-type and vcrD1 strains revealed that the mutant was defective in secretion of at least 19 proteins including several flagellar components. Western blot analyses using specific antibodies confirmed that the secretion of at least four flagellar components, such as FlaA, FlgL, FlgE, and FlgM, was affected by the vcrD1 mutation, which was consistent with decreased motility on soft agar plates and the non-flagellated morphology of the mutant. The exsA variant, another T3SS mutant, did not showed reduced motility, but exhibited a non-motile phenotype with the vcrD1 mutation. Complementation of wild-type vcrD1 gene into the vcrD1 mutant resulted in restored motility. Fractionation of bacterial cytoplasm from the periplasm and membrane revealed lower levels of FlaA and FlgM in the cytoplasm, indicating that VcrD1 might regulate the expression of flagellar genes in addition to the secretion of flagellar components in V. parahaemolyticus. Quantitative RT-PCR assays of seven representative flagellar genes in the wild-type and vcrD1 mutant strains demonstrated that transcript levels of two early flagellar genes, flaK and flaL, were not affected by vcrD1 mutation, whereas the middle and late flagellar genes were down-regulated by the vcrD1 mutant. This study raises the possibility that VcrD1 plays a role in flagellar morphogenesis in V. parahaemolyticus by regulating the expression and secretion of flagellar components.ope

Effect of high glucose on nitric oxide production in cultured rat mesangial cells

의학과/박사[한글] 당뇨병성 신증은 말기 신부전증의 주된 원인 질환으로 국내에서도 새로 진단 받은말기 신부전증 환자의 약 34%가 당뇨병성 신증에서 기인하는 것으로 보고되어 있다. 고포도당혈증은 당뇨병성 신증의 발생과 진행을 결정하는 주된 인자로 생각되고 있으나, 정확히 어떠한 원위 경로를 경유하여 사구체 모세혈관 기저막의 비후 및 메산지움 영역의 확장 등 특징적인 변화를 유발하는 지는 알려져 있지 않다. 고포도당혈증에 의하여 사구체 손상을 매개할 것으로 보고되어 있는 여러 인자들 중 하나인 nitric oxide (NO)는 세포질 내에서 nitric oxide synthase (NOS)에 의하여 합성된다. 신장 내에서 생성되는 consti-tutive NO는 사구체 모세혈관의 압력을 감소시키고 신장 혈류 순환의 자동 조절(autore-gulation) 기능에 관여하며 세뇨관에서의 sodium 재흡수를 억제하는 기능을 하는 것으로 보고되어 있어 신 손상에 대한 보호 작용을 하는 것으로 생각되고 있으나, 병적인 조건하에서 inducible NOS (iNOS) 경로에 의하여 국소적으로 다량의 NO가 생성되는 경우에는 오히려 신장의 손상을 유발할 수 있는 것으로 알려져 있다. 당뇨병성 신증의 발생에 있어서는 초기에 관찰되는 사구체의 과여과에 constitutive NOS (cNOS)에 의한 NO의 합성 증가가 관여하는 것으로 제시되어 있으며 당뇨병의 진행에 따라 NO의 안정성 및 활성이 감소되는 것으로 보고되어 있으나, iNOS 경로에 의한 NO의 역할에 대해서는 확실히 알려져 있지 않다. 이에 본 연구자는 백서에서 추출한 사구체 메산지움 세포를 배양하여 고포도당에 노출시킨 후 iNOS에 의한 NO의 합성 변화를 관찰하였으며, 고포도당에 의한 NO의 합성 변화가 궁극적으로 세포 외 기질의 합성에 미치는 영향을 평가하여 다음의 결과를 얻었다. 1. 고포도당에 노출된 메산지움 세포에서 배지 내 nitrite 및 nitrate의 총 농도는 lipo-polysaccharide (LPS)와 interferon-γ (IFN-γ)로 세포를 자극한 후 24시간째에 대조군에 비하여 의의있게 증가되었으며, NO에 의하여 이차적으로 활성화되는 세포 내 cyclic guanosine monophosphate의 농도도 자극 후 24시간 및 48시간째에 대조군에 비하여 의의있게 증가하였다. 2. LPS와 IFN-γ로 자극된 메산지움 세포의 iNOS mRNA 표현은 시간이 경과할수록 증가하였으며, 자극 후 4시간 및 24시간째에 메산지움 세포의 iNOS mRNA표현이 대조군에 비하여 의의있게 증가되었다. 그러나 동일 농도의 L형 포도당에 의해서는 iNOS mRNA의 표현이 증가하지 않았다. 3. LPS와 IFN-γ로 자극된 메산지움 세포의 iNOS 단백 표현은 시간이 경과할수록 증가하였으며, 자극 후 48시간째에는 대조군에 비하여 통계적으로 유의하게 증가되었다. 4. PKC 억제제 calphostin C에 의하여 고포도당에 의한 iNOS mRNA의 표현 증가가 억제되었다. 5. Aldose reductase 억제제에 의하여 고포도당에 의한 iNOS mRNA의 표현 증가가 억제되었다. 6. LPS와 IFN-γ로 자극된 메산지움 세포에서 고포도당에 의하여 fibronectin의 단백표현이 의의있게 증가하였으며, aminoguanidine을 이용하여 iNOS를 억제한 결과 고포도당에 의한 fibronectin 단백의 표현 증가가 의의있게 억제되었다. 본 연구의 결과로 백서 사구체 메산지움 세포에서 고포도당에 의하여 iNOS 경로에 의한 NO의 합성이 의의있게 증가됨을 확인하였으며, PKC 경로의 활성화와 polyol 대사의 항진이 이에 관여하는 것으로 사료된다. 또한 iNOS에 의한 NO의 합성 증가는 고포도당에 의한 메산지움 세포의 세포 외 기질 합성 증가를 촉진하는 원인이 될 수 있을 것으로 생각된다. [영문] Diabetic nephropathy is one of the leading causes of end-stage renal disease and characterized pathologically by the glomerular mesangial expansion and increased extracellular matrix (ECM) formation. Glomerular hyperfiltration and increased vascular permeability observed in the early stage of diabetic nephropathy have been proposed to play a significant pathophysiologic role in the eventual development of glomerulosclerosis of diabetic nephropathy. Some studies have suggested that this glomerular hyperfiltration is mediated by increased nitric oxide (NO) pro-duction via the constitutive nitric oxide synthase (cNOS) pathway present in endothelial cells under the high glucose environment. However, the exact role of the inducible NOS (iNOS) pathway present in mesangial cells in the pathogenesis of diabetic nephropathy is not clearly established. The present study was carried out to examine whether NO production via the iNOS pathway is modulated in cultured rat mesangial cells exposed to the high glucose environment and underlying mechanism of this modulation. Another aim of this study was to determine the role of NO in the increased production of ECM, induced by high glucose concentration. For this purpose, the production of the stable metabolite of NO (nitrite), intracellular cyclic guanosine monophosphate (cGMP), iNOS mRNA expression and iNOS protein synthesis were examined under different glucose concentrations. In addition, the effect of inhibition of the iNOS pathway on fibronectin synthesis was determined to examine the role of the iNOS pathway in high glucose-induced fibronectin synthesis by mesangial cells. Rat mesangial cells cultured in high glucose concentration (30 mM D-glucose) increased significantly nitrite/nitrate production and intracellular cGMP levels upon stimulation with lipopolysaccharide (LPS) plus interferon-γ(IFN-γ) compared with control glucose concentration (5.6 mM D-glucose). Mesangial iNOS mRNA expression and protein synthesis also increased significantly in response to high glucose. This enhanced iNOS mRNA expression induced by high glucose concentration was significantly suppressed by protein kinase C (PKC) inhibitor, calphostin C, and the aldose reductase inhibitor,6-bromo-1,3-dioxo-lH-benz[d,e]isoquinoline-2(3H)-acetic acid. High glucose also significantly increased fibronectin protein synthesis of mesangial cells compared to control glucose upon stimulation with LPS plus IFN- γ Amino-guanidine reversed this high glucose-induced fibronectin synthesis with inhibition of mesangial iNOS mRNA expression. These results indicate that high glucose in combination with stimulation by LPS plus IFN- γ enhances NO production from mesangial cells by the iNOS pathway, and that the activation of PKC and the polyol pathway may play a role in this enhancement. In addition, increased NO production by the iNOS pathway may promote extracellular matrix accumulation by mesangial cells under high glucose condition.restrictio

Comparison of the patterns of hyperlipidemia between idiopathic nephrotic syndrome and diabetic nephrotic syndrome

의학과/석사[한글] 신증후군 환자에서 고지혈증(hyperlipidemia) 및 고지단백혈증(hyperlipoproteinemia)은 흔히 동반되는 소견이다. 신증후군과 동반되는 고지혈증은 특징적으로 혈청 total cholesterol이 증가하고, 심한 단백뇨를 가지는 환자에 있어서 혈청 triglyceride의 증가를 동반할 수 있다 또한 apo-B를 지닌 지단백인 very low density lipoprotein(VLDL), intermediate density lipoprotein(IDL), low density lipoprotein(LDL)이 증가하는 반면 high density lipoprotein(HDL)은 정상이거나 증가 혹은 감소될 수 있는 것으로 알려져 있다. 지단백의 구성 자체에도 변화가 있어 지단백내의 cholesterol ester나 triglyceride의 양이 정상에 비하여 증가되어 있으며 구성하는 apoprotein의 아형이나 양에도 변화가 있는 것으로 알려져 있다. 신증후군에서 발생하는 고지혈증 및 고지단백혈증의 발생 기전에 대하여 많은 연구가 있었음에도 불구하고 아직까지 정확한 기전이 규명되어 있지 않다. 초기 연구자들은 원발성 사구체 신염에 의한 신증후군에서 고지혈증 및 고지단백혈증이 발생하는 주요 원인으 로 간에서의 apo-B 지단백의 생성 증가를 제시하였으나 이후의 많은 임상 연구와 동물 실험 결과 지단백의 생성 증가 뿐 아니라 혈중 제거율의 감소 또한 중요한 기전인 것으로 제시되었다. 당뇨병성 신증후군에서도 원발성 사구체 신염과 같은 유형, 같은 기전의 고 지혈증 및 고지단백혈증이 동반되는지에 관한 연구는 국내외적으로 매우 드문 실정으로 이를 알아보기 위하여 본 연구를 진행하였다. 본 연구는 1990년 1월부터 1995년 2월까지 연세대학교 의과대학 부속 세브란스 병원에 내원하여 신증후군으로 진단된 원발성 사구체 신염 환자와 인슐린비의존형 당뇨병성 신증후군 환자를 대상으로 후향적 연구에 의해 혈청 total cholesterol과 triglyceride, HDL-cholesterol을 조사하여 지질 분포의 유형을 비교하고, 아울러 혈청 total cholesterol의 증가량과 24시간 단백뇨, 혈청 알부민 및 혈청 알부민 감소량과의 상관관계를 분석하였다. 1. 총 대상 환자는 89예로 미세 변화형(minimal change disease, MCD)군이 45예, 비미세 변화형(non-MCD)군이 25예였으며 당뇨병성 신증후군이 18예였다. 각 군의 남녀비는 각각 30:15, 14:12, 7:11이었으며 평균 연령은 당뇨병성 신증후군에서 가장 높았다. Non-MCD군은 국소성 분절성 사구체 경화증(focal segmental glomerulosclerosis)이 5예, 막성 신증(membranous g1merulonephropathy)이 14예, 막 증식성 사구체 신염 (membranoproliferative g1omerulonephritis)이 4예, IgA 신증(IgA nephropathy)이 3예였다. 2. MCD, non-MCD, 당뇨병성 신증후군 모두에서 혈청 total cholesterol과 trig1yceride치가 증가되고 HDL-cholesterol치는 정상으로 지질 분포의 유형은 각 군 사이에 차이가 없었다. 3. 혈청 total cholesterol치는 MCD군에서 489.7±128.2 mg/dl, non-MCD군에서 410.3±158.2 mg/dl, 당뇨병성 신증후군에서 308.7±55.1 mg/dl로 MCD, non-MCD 환자에서 당뇨병성 신증후군 환자에 비해 의의있게 높았다(p＜0.05). 4. MCD, non-MCO군에서는 혈청 total cholesterol 증가량이 뇨단백 소실량과 유의하게 정상관관계가 있었으나(p＜O.05), 당뇨병성 신증후군에서는 이와 같은 정상관관계를 관찰할 수 없었고, 뇨단백 소실량에 대한 total cholesterol의 증가량도 MCD, non-MCD군에 비 해 의의있게 낮았다(p＜0.05). 5. 혈청 trilglyceride의 증가량은 MCD, non-MCD, 당뇨병성 신중후군 사이에 의의있는 차이가 없었다. 이상의 결과에서, 당뇨병성 신증후군에서는 원발성 사구체 신염에 의한 신증후군과 서로 다른 고지혈증 양상을 관찰할 수 있었다. 뇨단백 소실량에 대한 혈청 total cholesterol의 증가량이 당뇨병성 신증후군에서 원발성 사구체 신염보다 낮게 나타난 것은 간에서의 cholesterol의 생성 자체가 원발성 사구체 신염에 비하여 당뇨병성 신증후군에서 적을 가능성과 cholesterol의 생성 정도는 같으나 원발성 사구체 신염에서 apo-B 지단백의 혈중 제거율이 더욱 감소되어 있을 가능성, 그리고 이러한 두가지 기전의 복합적인 작용의 가능성을 생각할 수 있다. 향후 이러한 기전을 규명하기 위한 전향적 연구가 필요할 것으로 사료된다. Comparison of the patterns of hyperlipidemia between athic nephrotic syndrome and diabetic nephrotic syndrome Hyun Jin Noh Department of Medicine The Graduate School, Yonsei University (Directed by Professor Ho Yung Lee) Hyperlipidemia and hyperlipoproteinemia are major feature of the nerhrotic syndrome. An increased risk for premature coronary atherosclerosis has been nssociated strongly with elevated levels of total and low density lipoprotein(LDL)-cholesterol, depressed high density lipoprotein(HDL) -cholesterol, and high total to HDL-cholesterol ratio. These lipid and lipoprotein abnormalities commonly occurin ratients with nephrotic syndrome. The most common finding is an elevation of LDL-cholesterol levels. In more severe cases with heavy proteinuria, hyperlriglyceridemia and elevated very low density lipoprotein (VLDL)-cholesterol levels may become the dominant abnormalities. HDL-cholesterol levels have been variously recorded as high, low or normal. The mechanism for its occurrence is comp1ex and still controversial. For many year it has been postulated that the primary mechanism for hyperlipidemia in the nephrotic syndrome is a hepatic oveproduction and secretion of apo-B containing lipoproteins. Also it has been proposed that hypoalbuminemia induces the oversynthesis of lipoproteins. More recently, however, reduced catabolism of ape-B containing lipoproteins was suggested as an important mechanism for hyperlipidemia and hyperlipopoteinemia. Thus, two mechanisms might contribute to nephrotic dyslipidemia: an overproduction of apo-B containing lipoproteins and an impaired catabolism of these lipoproteins. Few studies were performed to determine whether nephrotic syndrome due to systemic disease such as DM(diabetes mellitus) and primary glomerulonehritis (GN) have similar or dissimilar patterns of dyslipidemia. I retrospectively reviewed the clinical records of patients with the nephrotic syndrome in Yonsei Medical Center from January 1, 1991 to Feburary 28, 1995. Serum tota1 cholesterol, triglyceride and HDL-cholesterol levels were assessed and the correlations among the increment of serum total cholesterol, 24 hour proteinuria, serum albumin and the decrement of serum albumin were analyzed. 1. Among 89 patients, 71 patients were primary GN and 18 patients were DM nephropathy. Of primary GN, 45 patients were minimal change disease(MCD) and 26 patients were non-MCD. In nun-MCD group, there were 5 focal segmental glomerulosclerosis (FSGS ), 14 membranous glomeu]onephropathy (MGN), 4 membranoproliferative glomerulonephritis (MPGN) and 3 IgA nephropathy patients. Mean age was oldest in DM nephropathy group. 2. In MCO. non-MCD and DM nephropathy groups, serum total chulesterol and triglyceride levels were increased and HDL-cholesterol levels were normal. 3. The serum total cholesterol levels were significantly higher in MCD and non-MCD groups than DM nephropathy group(489.7± 128.2, 410.3±l58.2, 308.7±55.1 mg/dl in MCD, non-MCD and DM nephropathy, respectively) (p＜0.05). 4. There was a significant direct correlation between the increment of serum total cholesterol and the decrement of serum albumin in MCD and non-MCD groups, but not in DM nephropathy group. And the increment of serum total cholesterol to the decrement of serum albumin ratio was significantly lower in DM nephropathy group than that in MCD and non-MCD groups. 5. The serum triglyceride levels were increased in MCD, non-MCD and DM nephropathy groups, and there were no significant differences among the groups. 6. In MCD, non-MCD and DM nephropathy groups, the amount of 24 hour urine protein and serum triglyceride levels were not different, so it was postulated that the amount of urinary losses of liporegulatory macrmolecules such as apo-C Ⅱ and heparan sulfate, known as lipoprotein lipase cofactors, were similar in three groups. These results suggest that relatively milder hypercholesterolemia in DM nephropathy group might be due to different mechanisms for dyslipidemia in DM nephropathy group from primary GN group. First, hepatic overproduction of cholesterol might be relatively mild in DM nephropathy group. Second, despite of similar degree of hepatic overproduction in two gruops, the removal of apo-B containing lipoproteins might be more impaired in primary GN group. And the combination of these two mechanisms might be another mechanism. Further studies will be necessary to clarify the different mechanisms by measuring the amount of urinary loss of macromolecules, the activities of 3-hydroxy -3- methylglutaryl- CoA-reductase(HMG-CoA-reductase), tumover rates of LDL-apo B, fractional catabolic rates of LDL-apo B and LDL input rates. [영문] Hyperlipidemia and hyperlipoproteinemia are major feature of the nerhrotic syndrome. An increased risk for premature coronary atherosclerosis has been nssociated strongly with elevated levels of total and low density lipoprotein(LDL)-cholesterol, depressed high density lipoprotein(HDL) -cholesterol, and high total to HDL-cholesterol ratio. These lipid and lipoprotein abnormalities commonly occurin ratients with nephrotic syndrome. The most common finding is an elevation of LDL-cholesterol levels. In more severe cases with heavy proteinuria, hyperlriglyceridemia and elevated very low density lipoprotein (VLDL)-cholesterol levels may become the dominant abnormalities. HDL-cholesterol levels have been variously recorded as high, low or normal. The mechanism for its occurrence is comp1ex and still controversial. For many year it has been postulated that the primary mechanism for hyperlipidemia in the nephrotic syndrome is a hepatic oveproduction and secretion of apo-B containing lipoproteins. Also it has been proposed that hypoalbuminemia induces the oversynthesis of lipoproteins. More recently, however, reduced catabolism of ape-B containing lipoproteins was suggested as an important mechanism for hyperlipidemia and hyperlipopoteinemia. Thus, two mechanisms might contribute to nephrotic dyslipidemia: an overproduction of apo-B containing lipoproteins and an impaired catabolism of these lipoproteins. Few studies were performed to determine whether nephrotic syndrome due to systemic disease such as DM(diabetes mellitus) and primary glomerulonehritis (GN) have similar or dissimilar patterns of dyslipidemia. I retrospectively reviewed the clinical records of patients with the nephrotic syndrome in Yonsei Medical Center from January 1, 1991 to Feburary 28, 1995. Serum tota1 cholesterol, triglyceride and HDL-cholesterol levels were assessed and the correlations among the increment of serum total cholesterol, 24 hour proteinuria, serum albumin and the decrement of serum albumin were analyzed. 1. Among 89 patients, 71 patients were primary GN and 18 patients were DM nephropathy. Of primary GN, 45 patients were minimal change disease(MCD) and 26 patients were non-MCD. In nun-MCD group, there were 5 focal segmental glomerulosclerosis (FSGS ), 14 membranous glomeu]onephropathy (MGN), 4 membranoproliferative glomerulonephritis (MPGN) and 3 IgA nephropathy patients. Mean age was oldest in DM nephropathy group. 2. In MCO. non-MCD and DM nephropathy groups, serum total chulesterol and triglyceride levels were increased and HDL-cholesterol levels were normal. 3. The serum total cholesterol levels were significantly higher in MCD and non-MCD groups than DM nephropathy group(489.7± 128.2, 410.3±l58.2, 308.7±55.1 mg/dl in MCD, non-MCD and DM nephropathy, respectively) (p＜0.05). 4. There was a significant direct correlation between the increment of serum total cholesterol and the decrement of serum albumin in MCD and non-MCD groups, but not in DM nephropathy group. And the increment of serum total cholesterol to the decrement of serum albumin ratio was significantly lower in DM nephropathy group than that in MCD and non-MCD groups. 5. The serum triglyceride levels were increased in MCD, non-MCD and DM nephropathy groups, and there were no significant differences among the groups. 6. In MCD, non-MCD and DM nephropathy groups, the amount of 24 hour urine protein and serum triglyceride levels were not different, so it was postulated that the amount of urinary losses of liporegulatory macrmolecules such as apo-C Ⅱ and heparan sulfate, known as lipoprotein lipase cofactors, were similar in three groups. These results suggest that relatively milder hypercholesterolemia in DM nephropathy group might be due to different mechanisms for dyslipidemia in DM nephropathy group from primary GN group. First, hepatic overproduction of cholesterol might be relatively mild in DM nephropathy group. Second, despite of similar degree of hepatic overproduction in two gruops, the removal of apo-B containing lipoproteins might be more impaired in primary GN group. And the combination of these two mechanisms might be another mechanism. Further studies will be necessary to clarify the different mechanisms by measuring the amount of urinary loss of macromolecules, the activities of 3-hydroxy -3- methylglutaryl- CoA-reductase(HMG-CoA-reductase), tumover rates of LDL-apo B, fractional catabolic rates of LDL-apo B and LDL input rates.restrictio

(The) sensitivity of pregnant rabbit uteri to epinephrine and norepinephrine and uterine catecholamine content

의학과/박사[한글] [영문] It has been known that norepinephrine and epinephrine, an neurohormonal transmitter, is synthesized and stored at the adrenergic nerve ending and that the uterus receives adrenergic terminals from the mesenteric ganglia. Many clinical studies to evaluate the action of these catecholamines in the pregnant uteri have been carried out since 1925 when Rucker first reported the diminished uterine contractions in the laboring patient by the use of epinephrine in combination with novocaine. Two years later, Bourne and Burn confirmed the fingind of decreased uterine contractions in labor following an intravenous drip of epinephrine, and Rucker also found that the injection of adrenaline causes a cessation of the uterine contractions and a relaxation of the constriction ring. Many investigators such as Rudolph and Ivy(1930), Ivy(1931), Irvins(1936), Titus(1937), Gunn(1942) and Garret(1954) have reported that epinephrine inhibits uterine contractions during pregnancy. On the contrary, Wooldbury, Hamilton and Torpin(1938) reported that the injection of epinephrine stimulates the uterine contraction. Woodbury and Abru(1944) stressed that the effect of epinephrine on the uterus is entirely depend upon its concentration; in low concentration the uterine contraction are inhibited whereas in high concentration they are simulated. Kaiser and Harris(1950) confirmed the findings of Woodbury in 140 pregnant women. Telko(1960) reported that coordinated contractions can be produced by injection of norepinephrine and seems to be more effective on uterine contractions can be produced by injection of norepinephrine and seems to be more effective on uterine contractions than oxytocin. In recent years, Pose et al. have reported that the intravunous infusion of 1- epinephrine in labor showed a decrease in the intensity of the uterine contractions, and infusion on the 1-norepinephrine in labor produced a clear increase in the contractility of the uterus and the induced contractility had a significantly different pattern from the spontaneous or oxytocin-induced ones. Cha(1965) ahs found that the catecholamine concentration of the uterus become reduced during pregnancy, particularly at the placental site, and a small amount of norepinephrine(10^^7 M) inhibits and a large amount stimulates the uterine activity wheres epinephrine always stimulates unterine ativity regardless the amount. It is clear that many controversial opinions exist about the effect of epinephrine and norepinephrine on the pregnant uterus and there seems to be some relation between uterine catecholamine content and pregnancy. Therefore, the present study was undertaken to investigate uterine catecholamine content and uterine sensitivity to epinephrine and norepinephrine during pregnancy. Materials and Methods The experiments were conducted on the adult, female, albino rebbits subdivided into groups of non-pregnant, postpartum and reserpine treated. In all groups, uterine strips were prepared from the placental site, non-placental site and intersegmental bath (38℃) containing 100 ml. of Locke's solution aerated with 95% oxygen and 5% carbon dioxide. Spontanuous motility was recorded with as isotonic lever. The epinephrine and norepinephrine concentrations of the uteri were determined by the method of Shore and Olin(1958). Reselts 1. Non-pregnant: The effect of epinephrine on the non-pregnant uterine segment, during estrus, was stimulative to the uterine tonus and the sivilar effect was also observed with the norepinephrine. The effect of epinephrine on the dibenamine-treated uterine segment was inhibitory whereas the dichloroisoproterenol-treated uterine segment showed increased contractility. Since the weight of the uterus varies with the preganacy, the total catecholamine content per uterus(mμg/uterus) could change even though the concentration of amines (mμg/g) could remain unaltered. For this reason, the amount of catecholamines per uterus and the amount per gram of uterus are both presented. The catecholamine concentration of the non-preganant uterus was 331 mμg/g measuring 131 mμg/g of epinephrine and 200 mμg/g of norepinephrine, and the totla uterine contents of epinephrine and norepinephrine were 520 mμg/uterus and 810 mμg/uterus respectively, The norepinephrine, both in concentration and in totla content pr uterus, showed higher amount than that of the epinephrine. 2. Pregnant: In the pregnant uterine segments the effects of epinephrine and norepinephrine varied according to the period of pregnancy. a) In early pregnancy (8-9 days), the uterine activity was inhibited by a low concentration of epinephrine or norepinephrine and, on the contrary, the uterine activity was stimulated by a higher concentration. The concentration of epinephrine and norepinephrine were 117 mμg/g and 60 mμg/g respectively and the total uterine contents were 931 mμg/uterus and 458 mμg/uterus respectively. The amount of norepinephrine becoame smaller than that of the epinephrine. b) In mid-pregnancy(17-18 days), the effect of catecholamine on the uterine activity ws greater than that in early pregnancy. The catecholamine concentration. however, was significantly decreased from the value of early pregnancy with marked reduction of the epinephrine. The epinephrine concentration was 46 mμg/g and norepinephrine was 51 mμg/g. The total uterine epinephrine content was 776 mμg/uterus, and total uterine norepinephrine was 846 mμg/uterus which was increased significantly c) In late pregnancy(26-28 days), the effect of catecholamine on the uterine activity was markedly increased. The catecholamine concentration was also slightly increased over the value of mid-pregnancy as 50 mμg/g of epinephrine and 69 mμg/g of norepinephrine, The total coatecholamine content in this stage, however, showed the highest value of all during pregnancy such as 1047 mμg/uterus for the total epinephrine content and 1437 mμg/uterus for the norepinephrine contents. 3. Post-partum: the uterus soon after the delivery, the sensitivity of all uterine segments to the catecholamines showed a pattern similar to the late pregnancy. But in 24 hours or more after the delivery the uterine segment demonstrated decreased actiity and a tendency to return to normal. The catecholamine concentration was slightly higher than in late pregnancy; 71 mμg/g of epinephrine and 83 mμg/g of norepinephrine, but the total uterine contents were less thn that of the late pregnancy as the total epinephrine content was 980 mμg/uterus and the total norepinephrine was 1160 mμg/uterus. 4. Reserpine-treated: The sensitivity of the uterine segments prepared from the late pregnancy rabbits treated with repserpine (3 mg/kg intraperitoneally 24 hours before sacrifice) to the catecholamines ws diminished in general. A stimulation effect did occur only by a higher concentration(more than 10^^6 M) of the catechloamines while an inhibitory action was observed by the lower concentration. This sensitivity showed a pattern similar to the early pregnancy. The uterine catecholamines in this group were markedly reduced, particularly so for the norepinephrine content. Summary The uterine catecholamine concentration which is present in considerably high level in normal uterus was started to decrease in the early pregnancy, reached the lowest level in the mid-pregnancy and then started to increase again in the late pregnancy when the total catecholamine content become the highest level of all. This increase of catecholamine in late pregnancy in late pregnancy was chiefly due to the increase of norepinephrine. On the other hand, the effect of catecholamine on the uterine activity ws first inhibitory in the early pregnancy but became gradually stimulative as the pregnancy progressed. This stimulating action on the uterine motility was found to occur through the action of norepinephrine. It is suggestive that uterine motility may be related to the catecholamine content, especially norepinephrine, in the uterus.restrictio

Study on RIPK3 protein expression and kinase activity in disease pathogenesis

학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2022. 8네크롭토시스는 분자적 기전을 통해 조절되는 세포 사멸 기작의 한 형태이다. 형태학적으로는 세포소기관의 팽창, 세포막의 파열 등 기존에 알려진 네크로시스의 특징을 보인다. 하지만 RIPK1, RIPK3, MLKL 등 주요 인자에 의해 세포 사멸이 특정한 기전을 따라 조절되는 양상이 네크로시스와 차별화된 특징이다. 네크롭토시스는 아폽토시스와 다르게 caspase에 비의존적인 기전을 가지며 결과적으로 세포막의 파열에 의한 세포 내 물질이 방출되기 때문에 주변의 면역 세포를 불러들임으로써 면역 반응을 유도하는 특징도 보인다. 네크롭토시스는 많은 보고를 통해 인간의 질병 발생 단계에 있어 관련성을 가진다고 알려져왔다. 주요 조절 인자인 RIPK3, MLKL 등의 knock out 기법을 이용하여 만든 동물 모델에서의 질병 관찰과 실제 환자의 티슈 분석을 통해 특히 RIPK3의 관련성이 중요한 의미를 띤다고 밝혀져왔다. 이러한 RIPK3의 발현과 활성을 조절하는 것이 질병 발생과의 연관성에 대한 연구를 할 때 중요한 점임을 시사한다. RIPK3는 세린/트레오닌 인산화 효소로 번역 후 수정 과정을 통해 그 활성이 조절된다고 알려진 단백질이다. 발현이 증가하거나 어떤 특정 상황에서 세린 227번 잔기에 자가인산화가 일어나면 인산화 효소로서 활성화된다. 하지만 어떠한 상황에서 어떻게 활성이 조절되는지에 대한 자세한 분자적 기전을 아직 밝혀지지 않았다. 이에 본 논문은 RIPK3의 활성을 조절하는 기전을 설명하고자 했다. PART Ⅰ은 관절염 모델에서 발현이 증가한 RIPK3의 기능에 대한 연구 결과를 보고한 것이며, PART Ⅱ는 활성산소에 의해 RIPK3가 자가인산화 되어 활성을 띠는 분자적 기전에 대한 연구 결과를 보고한 것이다. 현재까지 많은 질병에서 RIPK3의 역할이 보고되었지만 관절염 모델에서 RIPK3의 기능에 대한 연구는 거의 없었다. 이에 실제 관절염 환자의 조직을 분석하여 증가된 RIPK3의 발현을 확인하였고, 관절염을 유도한 마우스 모델과 콘트로사이트 세포를 사용해 발현이 증가된 RIPK3에 의해 관절염의 유도마커인 MMP3, MMP13, COX2 등의 증가를 관찰하였다. 이는 RIPK3에 의해 관절염 발생이 더 촉진될 수 있음을 시사한다. 이에 RIPK3의 활성을 조절하는 것이 관절염 치료제 개발에 있어 중요한 점이라 생각되어 RIPK3의 활성을 조절하는 약물로 AZ-628을 소개하였다. 결론적으로 발현이 증가된 RIPK3가 관절염을 촉진할 수 있으며, 이 때 RIPK3의 활성을 조절하는 AZ-628이 향후 관절염 관련 치료제로써 역할을 할 수 있는 가능성을 제시하였다. 두 번째 연구에서는 활성산소에 의해 시스테인 변형이 일어난 RIPK3가 자가인산화 되어 활성화되는 분자적 기전을 제시하였다. 세포 내에서는 대사 스트레스, 저산소증 등 활성산소에 대량 발생하는 상황이 관찰된다. 하지만 대부분의 경우 활성산소를 제거하는 기전으로 인해 세포 내 항상성이 유지되는데 이러한 항상성에 불균형이 생길 경우 활성산소는 RIPK3의 활성화에 기여할 수 있다. 활성산소는 시스테인 잔기의 산화를 유도하여 단백질 간 이황화 결합이 형성되는 원인으로작용한다. 이황화 결합이 형성된 단백질은 올리고머의 형태로 관찰되며 단백질 간 자가인산화 과정을 거쳐 인산화가 일어난다. 이러한 기전으로 RIPK3도 자가 인산화 되어 활성을 나타낸다. 본 연구를 통해 RIPK3의 활성이 어떻게 조절되는지에 대한 기전을 제시함으로써 활성산소에 의한 스트레스에 영향을 받는 다양한 질병에서 RIPK3의 역할에 대한 가능성을 시사하였다.IIntroduction 1 PART Ⅰ 4 TRIM24-RIPK3 axis perturbation accelerates osteoarthritis pathogenesis 4 I. Introduction 5 II. Materials and Methods 7 A. Human OA cartilage samples and experimental OA mouse models 7 B. Reagents 7 C. Primary mouse articular chondrocytes 8 D. Cell viability assay 9 E. Histology and immunohistochemistry 9 F. Western blotting 10 G. RT-PCR and qPCR 10 H. Collagenase and aggrecanase activity assay 10 I. Microarray analyses 11 J. In silico binding assay 11 K. Gene set enrichment analysis (GSEA) 12 L. Statistical analysis 12 III. Results 13 1. RIPK3 and MLKL expression patterns in various mouse tissue. 13 2. Non-canonical role of receptor-interacting protein kinase-3 (RIPK3) is related to osteoarthritis (OA) pathogenesis. 16 3. OA-signature genes were upregulated in RIPK3-overexpressing chondrocytes. 19 4. RIPK3 modulates OA pathogenesis 22 5. RIPK3 plays a key role in OA pathogenesis. 25 6. RIPK3 upregulation may potentiate OA pathogenesis via non-canonical MLKL-independent functions. 27 7. TRIM24 negatively regulates RIPK3-mediated OA pathogenic signatures 30 8. TRIM24 may negatively regulate elevated RIPK3 expression to accelerate OA pathogenesis 33 9. Identification of drugs that correlate with RIPK3 expression using CMap 36 10. RIPK3 kinase inhibition abrogates Ripk3 activity in OA pathogenesis 40 11. RIPK3 kinase activity inhibition by AZ-628 attenuated cartilage pathophysiology 43 IV. Discussion 45 PART Ⅱ 48 Regulation of ROS-mediated RIPK3 activation under glucose deprivation 48 I. Introduction 49 II. Materials and Methods 52 1. Cell lines and culture conditions 52 2. Antibodies and chemical reagents 52 3. Plasmid construction, mutagenesis and transfection 53 4. shRNA and reverse transcription-PCR (RT-PCR) 54 5. Purification of recombinant proteins 54 6. Cytotoxicity Assays 55 7. ROS measurement 55 8. Immunoblot analysis 56 9. Immunofluorescence analysis 56 10. Statistical analysis 57 III. Results 58 1. RIPK3 is phosphorylated at ser 227, followed by MLKL activation under glucose derivation condition 58 2. RIPK3 mediates MLKL phosphorylation in RIPK1-independent manner 61 3. AMPK is dispensable for RIPK3 activation under glucose deprivation condition 64 4. Accumulated ROS induce RIPK3 phosphorylation 67 5. ROS leads to RIPK1-independent MLKL phosphorylation 70 6. RIPK3 forms oligomerization through inter-chain disulfide bond 73 7. RIPK3 forms inter-chain disulfide bond dependent oligomerization during ROS induction 76 8. RIPK3 forms oligomerization in RHIM-independent manner 79 9. Activated MLKL accelerates cell death under glucose deprivation condition. 82 IV. Discussion 85 V. Reference 88 국문 요약 107DoctoralNecroptosis is a type of programmed cell death triggered by various stimuli including tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), interferon (IFN), and lipopolysaccharide (LPS). Multiple stimuli lead to necrosome formation, followed by activation of key mediators of necroptosis such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL) in a caspase-independent manner. Activated MLKL translocates to the plasma membrane, ultimately leading to cell rupture. Since released damage-associated molecular patterns (DAMPs) recruit immune cells and activate immune responses, necroptosis has recently been classified as an immunogenic cell death (ICD) response. Dysregulation of necroptosis has been known to be involved in numerous pathological conditions. Therefore, modulation of effector molecules, especially RIPK3, is thought to provide potential options for therapeutic intervention. Although the regulation of RIPK3 in TNF-mediated necroptosis is well defined, how RIPK3 is regulated under physiological conditions remains to be elucidated. In part Ⅰ of this study, the role of RIPK3 in osteoarthritis (OA), which is a degenerative joint disease, was investigated. Although necroptosis has recently been reported to drive arthritis, it remains unknown to what extent RIPK3 regulation is involved in OA. As RIPK3 plays a crucial role in necroptosis and dysregulation of RIPK3 is involved in various pathological processes, the role of the RIPK3 axis in OA pathogenesis was investigated. In vitro and in vivo analyses demonstrated that overexpression of RIPK3 accelerated cartilage disruption, whereas depletion of Ripk3 reduced DMM-induced OA pathogenesis. Furthermore, knockdown of tripartite motif containing 24 (TRIM24) upregulated RIPK3 expression and its downregulation promoted OA pathogenesis in knee joint tissues. The potent RIPK3 inhibitor AZ-628 was identified by CMap approach and in silico binding assay, and inhibition of RIPK3 kinase activity abolished RIPK3-mediated OA pathogenesis. In part Ⅱ of this study, the mechanism regulating RIPK3 activity was defined. ROS-mediated RIPK3 phosphorylation occurs under conditions of glucose deprivation leading to oxidative stress. RIPK3 undergoes oxidation at cysteine residues and forms oligomers via inter-chain disulfide bonds in a RIPK homotypic interaction motif (RHIM)-domain independent manner. By defining how RIPK3 is regulated under physiological conditions, this study highlights the potential for the development of anti-tumor agents that produce ROS and that allow exploitation of necroptosis for therapeutic applications

Minimally invasive adenomyomectomy via a laparoscopic-assisted approach compared to a laparoscopic or laparotomic approach

Objective The present study assessed the safety and benefits of laparoscopic-assisted adenomyomectomy compared to laparoscopic or laparotomic adenomyomectomy. Materials and methods This study was a retrospective comparative study. A total of 277 patients underwent adenomyomectomy between January 2016 and January 2019 at the Department of Obstetrics and Gynaecology, Ulsan University Hospital, including 25 with laparoscopic-assisted adenomyomectomy, 82 with laparoscopic adenomyomectomy, and 170 with laparotomic adenomyomectomy. Laparoscopic-assisted adenomyomectomy consisted of a laparoscopic uterine artery procedure to reduce blood loss and a minimal incisional for laparotomic adenomyomectomy. An additional laparoscopic surgery was performed for possible pelvic pathology. Results Data on patient demographics, surgical indications, operative times, estimated blood loss (EBL), short-term complications, and postoperative hospital stays were compared. The laparoscopic-assisted surgery (LAS) and laparotomic groups were comparable in average EBL (208.0 ± 128.8 vs. 193.6 ± 193.0 ml, p = 0.11), weight of removed mass (85.5 ± 71.7 vs. 108.2 ± 91.9 g, p = 0.39), and postoperative hospital days (HDs) (4.5 ± 1.0 vs. 4.7 ± 0.8 days, p = 0.27). These values were lower in the laparoscopic group (EBL 119.5 ± 79.6 ml, mass weight 39.3 ± 25.9 g, HD 3.6 ± 0.8 days). Additional procedures, including myomectomy and combined severe endometriosis surgery, were more frequently performed in the LAS group than the laparotomic group. The mean operating time was longer in the LAS group (179.8 ± 36.6 min) than the other groups (laparoscopy 99.9 ± 40.6 min, p < 0.00; laparotomy 133.0 ± 41.1 min, p < 0.00). The three groups did not differ significantly in transfusion rates, hemoglobin changes, or perioperative complications. However, febrile morbidity was lower in the laparoscopic group than the LAS and laparotomic groups. Conclusion LAS adenomyomectomy allows for maximal debulking of adenomyosis via extracorporeal and intracorporeal procedures while retaining the advantages of the laparoscopic approach. Additional pelvic surgery for benign uterine and adnexal pathology may easily be performed with this approach

Prognostic factors of oncologic outcomes after fertility-preservative management with progestin in early-stage of endometrial cancer

Background: The aim of this study was to evaluate efficacy of various fertility-preservative treatments with progestin and analyze prognostic factors in Stage 1A of endometrial cancer. Materials and Methods: This retrospective study involved four Korean university hospitals. Data were collected from 43 women who were under the age of 40 with presumed stage IA endometrial cancer determined by magnetic resonance imaging and treated from January 2014 to December 2017. All of the patients were administered hormonal therapy for fertility preservation. Twenty-five patients received oral progestin with a levonorgestrel-releasing intrauterine system (LNG-IUS) for 6-24 months, and 18 patients received high-dose oral progestin for the same period of time. Oncologic outcomes were evaluated. Prognostic factors for pathologic response to progestin were identified by logistic regression analysis. Results: Complete response (CR) was achieved by 72.1% of patients (31/43), and the average time to CR was 4.2 (Stable disease [SD] 3.4) months (range, 3-9 months). Partial response was achieved by 7.0% of patients (3/43), SD by 9.3% (4/43), and progressive disease by 11.6% (5/43). Of the CR patients, 41.9% (13/31) achieved pregnancy with the median follow-up period of 12.5 (SD 7.6) months (range: 3-50 months). No irreversible toxicity or therapy-associated death occurred. Multivariate analysis showed that high endometrial thickness ratio of pre- and posttreatment measured at 2 months from the treatment initiation (>= 0.55, Odds ratio [OR]: 19.018; 95% confidence intervals (CI): 1.854-195.078; P = 0.013) and oral progestin without LNG-IUS (OR: 13.483; 95% CI: 1.356-134.069; P = 0.026) might be related with unfavorable prognostic factors for CR. Conclusion: This study shows that progestin-based fertility-preservative treatment might be a feasible option for stage 1A endometrial cancer. It also identifies that low endometrial thickness ratio and oral progestin with LNG-IUS combination therapy might be related with favorable response to hormonal treatment

Maternal Bochdalek Hernia during Pregnancy: A Systematic Review of Case Reports

Background: Since the first report of a diaphragmatic hernia from Ambroise Pare’s necropsy in 1610, the Bochdalek hernia (BH) of the congenital diaphragmatic hernia (CDH) has been the most common types with high morbidity and mortality in the neonatal period. Due to the nature of the disease, CDH associated with pregnancy is too infrequent to warrant reporting in the literature. Mortality of obstruction or strangulation is mostly due to failure to diagnose symptoms early. Early diagnosis and surgical intervention are imperative, as a gangrenous or non-viable bowel resection significantly increases mortality. Therefore, multidisciplinary care should be required in maternal BH during pregnancies that undergo surgically repair, and individualized care allow for optimal results for the mother and fetus