Involvement_of_Rho_Kinase_in_Endothelial__Barrier_Maintenance
Rho激酶抑制剂对大鼠血管内皮的保护作用及eNOS表达的影响
Rho激酶抑制剂对大鼠血管内皮的保护作用及eNOS表达的影响吴巧;马康华;张晓刚;董倩;赖晓峰【摘要】Objective To explore the protective effects of Rho kinase inhibitor (fasudil) on rats1 vascular endothelial cells and the effects on the expression of endothelial nitric oxide synthase (eNOS), Methods Thirty male SD rats were randomly divided into S groups (6 each): control group (intraperitoneal injection with 0.9% normal saline), hyperhomocysteinemia (HHcy) group; low-dose fasudil group [L-treatment group, intraperitoneal injection with lmg/(kg.d) fasudil], middle-dose fasudil group [M-treatment group, intraperitoneal injection with 5mg/(kg.d) fasudil], and high-dose fasudil group [H-treatment group, intraperitoneal injection with 15mg/(kg-d) fasudil]. Animals in HHcy group and fasudil groups were administered continuously with water containing 1.5% methionine for 4 weeks to establish HHcy damaged vascular endothelium model, and those in control group were only fed drinking water. After successful reproduction of model, the enzymatic method was applied to measure the serum level of nitric oxide (NO). The expressions of eNOS, Rho-associated coiled-coil protein kinase 2 (ROCK2) and RhoA protein tn aorta were assessed by immunohistochemistry and Western blotting. Results Compared with control group; the serum NO level and expression of eNOS protein in aorta decreased significantly in HHcy group (P<0.05), Compared with HHcy group, the serum NO level increased significantly in M- and H-treatmentgroup (P<0.05), but no statistical difference was found in L-treatment group (P>0.05). The aortic endothelial eNOS positive cells increased significantly in H-treatment group compared with that in HHcy group and L-treatment group (P<0.05), but no significant increase in L-treatment group as compared with that in HHcy group (P>0.05). Compared with HHcy group, the expressions of RhoA and ROCK2 decreased significantly in H-treatment group (P<0.05), but no significant change was found in L- and M-treatment group (P>0.05). Conclusions High-dose fasudil can protect vascular endothelia by inhibiting Rho/ROCK pathway to increase the expression of eNOS and NO.%目的探讨Rho激酶抑制剂法舒地尔对大鼠血管内皮的保护作用及内皮型一氧化氮合酶(eNOS)表达的影响.方法 30只雄性SD 大鼠随机分为5组(每组6只),即空白对照组(Control组,腹腔注射0.9%生理盐水),高同型半胱氨酸血症(HHcy)模型组(HHcy组,腹腔注射0.9%生理盐水),低、中、高剂量法舒地尔干预组[L-、M-、H-干预组,分别经腹腔注射法舒地尔l、5、15mg/(kg·d)].HHcy组及法舒地尔干预组均采用1.5%蛋氨酸饮用水持续饲养大鼠4周,构建HHcy血管内皮损伤模型;空白对照组大鼠以饮用水喂养.建模成功后采用酶法检测大鼠血清一氧化氮(NO)含量;分别用免疫组织化学法和Western blotting检测主动脉中eNOS、Rho相关的卷曲蛋白激酶(ROCK2)和Ras基因家族成员A(RhoA)蛋白的表达.结果与空白对照组比较,HHcy组大鼠血清中NO含量明显下降,主动脉中eNOS蛋白表达也明显下降(P<0.05).与HHcy组相比,M-干预组和H-干预组大鼠血清NO浓度明显升高,差异有统计学意义(P<0.05),而L-干预组则无明显变化(P>0.05); H-干预组主动脉血管内皮细胞eNOS表达明显高于HHcy组和L-干预组,差异有统计学意义(P<0.05).与空白对照组相比,HHcy组RhoA和ROCK2表达明显增高,差异有统计学意义(P<0.05);与HHcy组比较,H-干预组RhoA和ROCK2表达明显降低(P<0.05),而L-干预组和M-干预组则无明显差异(P>0.05).结论高剂量法舒地尔可能通过抑制Rho/Rho激酶信号通路,增加NO和eNOS的表达,发挥对大鼠血管内皮的保护作用.【期刊名称】《解放军医学杂志》【年(卷),期】2012(037)011【总页数】6页(P1044-1049)【关键词】内皮,血管;Rho/ROCK信号通路;法舒地尔;一氧化氮;内皮型一氧化氮合酶【作者】吴巧;马康华;张晓刚;董倩;赖晓峰【作者单位】400016 重庆重庆医科大学附属第一医院心内科;400016 重庆重庆医科大学附属第一医院心内科;400016 重庆重庆医科大学附属第一医院心内科;400016 重庆重庆医科大学附属第一医院心内科;400016 重庆重庆医科大学附属第一医院心内科【正文语种】中文【中图分类】R5411976年,Harker等[1]提出了内皮细胞损伤学说,后来又增加了“内皮细胞过度反应导致损伤”的概念。
氢键的应用
how the standard perception of halogen substituents, which assumes an isotropic negative electron density around the halogen, was replaced by a description that takes the σ-hole into account. Halogen bonds have been found to occur in a multitude of inorganic, organic, and biological systems.4,5 In an early study from the 1950s, Hassel and Hvoslef solved the crystal structure of the equimolar Br2:dioxane adduct and found Br···O contacts featuring distances substantially below the sum of the van der Waals radii of both atoms, indicating a strong attractive interaction between both atoms.6,7 In 1984, a search of the Cambridge crystallographic data files for short iodine···N/O/S contacts revealed that these interactions are also formed in biologically relevant systems, being employed by nature for the molecular recognition of thyroid hormones at their target proteins such as transthyretin.8 In protein−ligand environments, halogen bonds can be formed between a halogenated ligand and any accessible Lewis base in the binding pocket.9 Probably because of its presence in every amino acid, the backbone carbonyl oxygen function is the most prominent Lewis base involved in halogen bonds in protein binding sites, as found from an analysis of the Protein Data Bank (PDB).10,11 Additionally, halogen bonds can be formed involving side chain groups, such as hydroxyls in serine, threonine, and tyrosine, carboxylate groups in aspartate and glutamate, sulfurs in cysteine and methionine, nitrogens in histidine, and the π surfaces of phenylalanine, tyrosine, histidine, and tryptophan. Several examples for these contacts are given in Figure 2.
火龙罐疗法联合常规治疗在AECOPD_患者中的应用效果
- 143 -*基金项目:广东省中医药局立项项目(202105211511578370)①梅州市中医医院 广东 梅州 510000火龙罐疗法联合常规治疗在AECOPD患者中的应用效果*韩文聪① 陈晓英① 郑小芬① 邹爱萍①【摘要】 目的:探讨火龙罐疗法联合常规治疗在慢性阻塞性肺疾病急性加重期(AECOPD)患者中的应用效果。
方法:选取2022年1—10月在梅州市中医医院住院的100例AECOPD 患者作为研究对象,根据临床实际情况以及患者意愿,且按是否接受火龙罐治疗分为治疗组与对照组,各50例。
对照组给予常规治疗,治疗组给予火龙罐疗法联合常规治疗。
比较两组治疗前后症状积分、血气指标[动脉血氧分压(PaO 2)、动脉血二氧化碳分压(PaCO 2)],治疗前及随访3个月、6个月生活质量(ADL)与6分钟步行试验(6MWT)变化,不良反应发生情况。
结果:治疗后,两组症状积分、PaCO 2较治疗前降低,且治疗组低于对照组,两组PaO 2较治疗前提高,且治疗组高于对照组,差异有统计学意义(P <0.05)。
随访3个月、6个月,两组ADL 评分、6MWT 距离较治疗前提高,随访6个月两组各指标较随访3个月提高,且治疗组高于对照组,差异有统计学意义(P <0.05)。
两组治疗期间不良反应发生率比较,差异无统计学意义(P >0.05)。
结论:火龙罐疗法联合常规治疗AECOPD 效果满意,可缓解患者的临床症状,改善血气指标,提高生活质量及步行能力,同时不增加不良反应发生率。
【关键词】 慢性阻塞性肺疾病急性加重期 火龙罐疗法 康复 血气指标 doi:10.14033/ki.cfmr.2024.06.036 文献标识码 B 文章编号 1674-6805(2024)06-0143-04 Application Effect of Fire Dragon Cup Therapy Combined with Conventional Treatment in Patients with AECOPD/HAN Wencong, CHEN Xiaoying, ZHENG Xiaofen, ZOU Aiping. //Chinese and Foreign Medical Research, 2024, 22(6): 143-146 [Abstract] Objective: To investigate the application effect of Fire Dragon Cup therapy combined with conventional treatment in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Method: A total of 100 patients with AECOPD who hospitalized in Meizhou Traditional Chinese Medicine Hospital from January to October 2022 were selected as the research objects, according to the actual clinical situation and patients' wishes, they were divided into the treatment group and the control group according to whether they received Fire Dragon Cup treatment, with 50 cases in each group. The control group was given conventional treatment, and the treatment group was given Fire Dragon Cup therapy combined with conventional treatment. The symptom scores, blood gas indexes [arterial partial pressure of oxygen (PaO 2), arterial partial pressure of carbon dioxide (PaCO 2)] before and after treatment, quality of life (ADL) and 6-minute walking test (6 MWT) before treatment, 3 months and 6 months after follow-up, and incidence of adverse reactions were compared between two groups. Result: After treatment, the symptom scores and PaCO 2 of two groups were lower than those before 弹力纤维降解的影响及机制[J].中华实用诊断与治疗杂志,2020,34(1):9-12.[19] SAN NORBERTO E M,REVILLA Á,Fernandez-Urbon A,et al.Vascular calcification progression in patients with end-stage chronic kidney disease[J]. Int Angiol,2021,40(6):528-536.[20] LI H,YANG M. Ligustrazine activate the PPAR-γ pathwayand play a protective role in vascular calcification[J]. Vascular,2022,30(6):1224-1231.[21] WING T T,ERIKSON D W,BURGHARDT R C,et al. OPNbinds alpha V integrin to promote endothelial progenitor cell incorporation into vasculature[J]. Reproduction,2020,159(4):465-478.[22] OGATA H,FUKAGAWA M,HIRAKATA H,et al. Effect oftreating hyperphosphatemia with lanthanum carbonate vs calcium carbonate on cardiovascular events in patients with chronic kidney disease undergoing hemodialysis: the LANDMARK randomized clinical trial[J]. JAMA,2021,325(19):1946-1954.[23] SHIRAKAWA K,SANO M. Osteopontin in cardiovasculardiseases[J]. Biomolecules,2021,11(7):1047.[24] DE VRIESE A S,CALUW É R,PYFFEROEN L,et al.Multicenter randomized controlled trial of Vitamin K antagonist replacement by Rivaroxaban with or without Vitamin K 2 in hemodialysis patients with atrial fibrillation: the Valkyrie Study[J]. J Am Soc Nephrol,2020,31(1):186.(收稿日期:2023-08-22) 慢性阻塞性肺疾病(COPD)属于常见的一种慢性气道疾病,COPD分为稳定期与急性加重期[1]。
医学国际会议学术交流壁报模板
phosphoJNK
stimulated in vitro with agonists, and exposed for identical total periods to experimental
total JNK
conditions. Phospho-JNK was assessed by
phospho-JNK (% of unstimulated) phospho-JNK (% of unstimulated)
Aim
To investigate whether JNK participates in a1-adrenergic contraction of human prostate smooth muscle.
Methods
• Human prostatic tissue: radical prostatectomy
Figure 2: Effect of the JNK inhibitor SP600125 on electric field stimulation- (EFS-) induced
contraction of human prostate strips. Contraction of prostate tissue in response to EFS was determined by myographic measurements. SP200125 (50 mM) or the solvent DMSO were added between two cycles of EFS (30 minutes before the second cycle). Shown are frequencydependent concentrations. Data are means±SEM from experiments with tissues from n=7 patients. *, p<0.05 for DMSO vs. SP600125.
Rho激酶及其抑制剂的研究进展
Rho 激酶及其抑制剂的研究进展段为钢1,袁胜涛2,廖 红1,严 明1,张陆勇1*(中国药科大学1.新药筛选中心,2.江苏省药效研究与评价服务中心,江苏南京210009)摘要:Rho 激酶是近十年来发现参与细胞运动的主要激酶之一,对细胞的分裂、收缩、粘附、迁移、分泌等活动具有重要调节作用。
Rho 激酶的高表达或过度激活与许多心脑血管疾病的发生发展密切相关,R ho 激酶现在已经成为新药研发的重要靶点,而R ho 激酶抑制剂的不断发现为心血管、神经系统等疾病的治疗提供了新的希望。
为此,本文就Rho 激酶及其抑制剂的研究做一简要综述。
关键词:R ho 激酶;抑制剂;新药筛选中图分类号:R 916 文献标识码:A 文章编号:0513-4870(2007)10-1013-10收稿日期:2007-05-17.基金项目:国家科技部/十五0重大计划资助项目(2004AA2Z3785);江苏省教育厅/江苏省研究生培养创新工程0(02705024);教育部新世纪优秀人才支持计划.*通讯作者 T e:l 86-25-85391036,Fax :86-25-85303260,E-m a i :l drugscreen @126.co mAdvances i n t he study of Rho ki nase and its i nhi bitorsDUAN W e-i gang 1,YUAN Sheng -tao 2,LI A O Hong 1,YAN M ing 1,Z HANG Lu -yong1*(1.N e w D rug Screeni ng Center, 2.J iang su Center for Pharmacodynam ics R esearch and Evaluation ,China Pharmaceutical Uni versity,N anj i ng 210009,China )Abstract :Rho kinase ,a lso na m ed Rho associated kinase ,is one of the i m portant k i n ases found i nrecent ten years ,wh ich regu lates cell m ove m ent i n clud i n g cy tod i e resis ,con traction ,adherence ,m igrati o n,secretion ,etc .The Rho kinase up -regulation in activ ity or i n expressi o n i n vo l v es the prog ress o f card i o -cerebr o -vascu lar disor ders ,and Rho k i n ase has been regarded as a key tar get in drug discovery and deve l o pm ent .W ith m ore and m ore Rho kinase i n h i b itors popping up ,Rho k i n ase i n h i b itors are beco m ing a pr o m i s ing so l u ti o n to car d i o vascu lar d iseases ,neural disorders and other d iseases .The artic le rev ie w s the advances i n the study o f Rho k i n ase pathw ay and its inhibitors ,other i n for m ation associated w ith Rho kinase is a lso discussed .K ey w ords :Rho assoc iated k i n ase ;i n h i b itor ;dr ug screening Rho 激酶(Rho assoc i a ted kinase ,ROCK ),是参与细胞有丝分裂粘附、细胞骨架调整、肌肉细胞收缩、肿瘤细胞浸润等一系列细胞生命现象的重要酶[1]。
恩格列净联合二甲双胍治疗2型糖尿病的效果评价
恩格列净联合二甲双胍治疗2型糖尿病的效果评价林智化厦门大学附属第一医院同安院区(厦门市第三医院)内分泌科,福建厦门361100[摘要]目的分析恩格列净联合二甲双胍治疗2型糖尿病的效果。
方法选取2021年3月—2022年4月厦门大学附属第一医院同安院区(厦门市第三医院)收治的98例2型糖尿病患者为研究对象,按照随机数表法分为观察组和对照组,每组49例。
两组均予以二甲双胍治疗,同时观察组加行恩格列净治疗。
比较两组血糖水平、血脂水平、胰岛β细胞功能、不良反应发生率以及临床疗效。
结果治疗6个月后,与对照组相比,观察组血糖水平、血脂水平均较低,胰岛β细胞功能的改善情况较好,差异有统计学意义(P<0.05)。
观察组不良反应发生率8.16%与对照组6.12%对比,差异无统计学意义(P>0.05)。
观察组治疗总有效率95.92%较对照组83.67%更高,差异有统计学意义(P<0.05)。
结论应用恩格列净联合二甲双胍治疗,效果突出,可有效调节糖脂代谢,纠正胰岛β细胞功能。
[关键词] 恩格列净;二甲双胍;2型糖尿病;糖脂代谢;胰岛β细胞功能[中图分类号] R446.1 [文献标识码] A [文章编号] 1672-4062(2023)08(a)-0087-04 Efficacy Evaluation of Empagliflozin Combined with Metformin in the Treatment of Type 2 Diabetes MellitusLIN ZhihuaDepartment of Endocrinology, Tong'an District of the First Affiliated Hospital of Xiamen University (Xiamen Third Hospital), Xiamen, Fujian Province, 361100 China[Abstract] Objective To analyze the effect of Empagliflozin combined with metformin in the treatment of type 2 dia⁃betes mellitus. Methods From March 2021 to April 2022, 98 patients with type 2 diabetes treated in Tong 'an District the First Affiliated Hospital of Xiamen University (Xiamen Third Hospital) were selected as the research objects. Ac⁃cording to the random number table method, they were divided into observation group and control group, 49 cases in each group. Both groups were treated with metformin, while the observation group was treated with empagliflozin. The blood glucose level, blood lipid level, islet β cell function, incidence of adverse reactions and clinical efficacy were compared between the two groups. Results After 6 months of treatment, compared with the control group, the blood glucose level and blood lipid level in the observation group were lower, and the improvement of islet β cell function was better, the difference was statistically significant (P<0.05). There was no statistically significant difference in the incidence of adverse reactions between the observation group (8.16%) and the control group (6.12%) (P>0.05). The to⁃tal effective rate of treatment in the observation group was 95.92% higher than that in the control group (83.67%), and the difference was statistically significant (P<0.05). Conclusion The treatment of Empagliflozin combined with metfor⁃min has a remarkable effect, which can effectively regulate the metabolism of glucose and lipid and correct the func⁃tion of islet beta cells.[Key words] Empagliflozin; Metformin; Type 2 diabetes; Glycolipid metabolism; Islet beta cell function2型糖尿病是最为常见的糖尿病类型,由于多发于成年,故又称成人发病型糖尿病,疾病早期症状不典型,随着疾病进展,患者可出现多饮、多食、多尿、消瘦或短期内体质量减轻等典型症状[1]。
国际贸易英文版教材
作者、书名、出版社、出版年份、目录Thomas A.Pugel. International Economics(15th). Renmin University of China p ress. 2012-12CONTENTSChapter 1 International Economics Is DifferentFour ControversiesEconomics and the Nation-StateThe Scheme of This BookPART ONE THE THEORY OF INTERNATIONAL TRADEChapter 2 The Basic Theory Using Demand and SupplyFour Questions about TradeA Look AheadDemand and SupplyCase Study Trade Is ImportantGlobal Crisis The Trade Mini-Collapse of 2009Two National Markets and the Opening of TradeChapter 3 Why Everybody Trades: Comparative Advantage 33Adam Smith’s Theory of Absolute AdvantageCase Study Mercantilism: Older Than Smith—and Alive TodayRicardo’s Theory of Comparative AdvantageRicardo’s Constant Costs and the Producti on-Possibility CurveFocus on Labor Absolute Advantage Does MatterExtension What If Trade Doesn’t Balance?Chapter 4 Trade: Factor Availability and Factor Proportions Are KeyProduction with Increasing Marginal CostsCommunity Indifference CurvesProduction and Consumption TogetherFocus on China The Opening of Trade and China’s Shift Out of AgricultureThe Gains from TradeTrade Affects Production and ConsumptionWhat Determines the Trade Pattern?The Heckscher–Ohlin (H–O) TheoryChapter 5 Who Gains and Who Loses from Trade?Who Gains and Who Loses within a CountryThree Implications of the H–O TheoryExtension A Factor-Ratio ParadoxDoes Heckscher–Ohlin Explain Actual Trade Patterns?Case Study The Leontief ParadoxWhat Are the Export-Oriented and Import-Competing Factors?Focus on China China’s Exports and ImportsDo Factor Prices Equalize Internationally?Focus on Labor U.S. Jobs and Foreign Trade 86Chapter 6 Scale Economies, Imperfect Competition, and TradeScale EconomiesIntra-Industry TradeMonopolistic Competition and TradeExtension The Individual Firm in MonopolisticOligopoly and TradeExtension The Gravity Model of TradeChapter 7 Growth and TradeBalanced versus Biased GrowthGrowth in Only One FactorChanges in the Country’s Willingness to TradeCase Study The Dutch Disease and DeindustrializationEffects on the Country’s Terms of TradeTechnology and TradeFocus on Labor Trade, Technology, and U.S. WagesPART TWO TRADE POLICYChapter 8 Analysis of a TariffGlobal Governance WTO and GATT: Tariff SuccessA Preview of ConclusionsThe Effect of a Tariff on Domestic ProducersThe Effect of a Tariff on Domestic ConsumersThe Tariff as Government RevenueThe Net National Loss from a TariffExtension The Effective Rate of ProtectionCase Study They Tax Exports, TooThe Terms-of-Trade Effect and a Nationally Optimal TariffChapter 9 Nontariff Barriers to ImportsTypes of Nontariff Barriers to ImportsThe Import QuotaGlobal Governance The WTO: Beyond TariffsGlobal Crisis Dodging ProtectionismExtension A Domestic Monopoly Prefers a QuotaVoluntary Export Restraints (VERs)Other Nontariff BarriersCase Study VERs: Two ExamplesCase Study Carrots Are Fruit, Snails Are Fish, and X-Men Are Not HumansHow Big Are the Costs of Protection?International Trade DisputesFocus on China China’s First Decade in the WTOChapter 10 Arguments for and against ProtectionThe Ideal World of First BestThe Realistic World of Second BestPromoting Domestic Production or EmploymentThe Infant Industry ArgumentFocus on Labor How Much Does It Cost to Protect a Job?The Dying Industry Argument and Adjustment AssistanceThe Developing Government (Public Revenue) ArgumentOther Arguments for Protection: Non=economic ObjectivesThe Politics of Protection The Basic Elements of the Political-Economic Analysis Case Study How Sweet It Is (or Isn’t)Chapter 11 Pushing ExportsDumpingReacting to Dumping: What Should a Dumpee Think?Actual Antidumping Policies: What Is Unfair?Case Study Antidumping in ActionProposals for ReformExport SubsidiesWTO Rules on SubsidiesShould the Importing Country Impose Countervailing Duties?Case Study Agriculture Is AmazingStrategic Export Subsidies Could Be GoodGlobal Governance Dogfight at the WTOChapter 12 Trade Blocs and Trade BlocksTypes of Economic BlocsIs Trade Discrimination Good or Bad?The Basic Theory of Trade Blocs: Trade Creation and Trade DiversionOther Possible Gains from a Trade BlocThe EU ExperienceCase Study Postwar Trade Integration in EuropeNorth America Becomes a BlocTrade Blocs among Developing CountriesTrade EmbargoesChapter 13 Trade and the EnvironmentIs Free Trade Anti-Environment?Is the WTO Anti-Environment?Global Governance Dolphins, Turtles, and the WTOThe Specificity Rule AgainA Preview of Policy PrescriptionsTrade and Domestic PollutionTrans-border PollutionGlobal Environmental ChallengesChapter 14 Trade Policies for Developing CountriesWhich Trade Policy for Developing Countries?Are the Long-Run Price Trends against Primary Producers?Case Study Special Challenges of TransitionInternational Cartels to Raise Primary-Product PricesImport-Substituting Industrialization (ISI)Exports of Manufactures to Industrial CountriesChapter 15 Multinationals and Migration: International Factor MovementsForeign Direct InvestmentMultinational EnterprisesFDI: History and Current PatternsWhy Do Multinational Enterprises Exist?Taxation of Mul tinational Enterprises’ProfitsCase Study CEMEX: A Model Multinational from an Unusual PlaceMNEs and International TradeShould the Home Country Restrict FDI Outflows?Should the Host Country Restrict FDI Inflows?Focus on China China as a Host CountryMigrationHow Migration Affects Labor MarketsShould the Sending Country Restrict Emigration?Should the Receiving Country Restrict Immigration?Case Study Are Immigrants a Fiscal Burden?APPENDIXESA The Web and the Library: International Numbers and Other InformationB Deriving Production-Possibility CurvesC Offer CurvesD The Nationally Optimal Tariff周瑞琪. International Trade Practice. University of International Business and Economics press. 2011.9CONTENTSChapter One General Introduction(第一章导论)1.1 Reasons for International Trade (国际间贸易的起因)1.2 Differences between International Trade and Domestic Trade (国际贸易与国内贸易的差异)1.3 Classification of International Trade(国际贸易的分类)1.4 Export and Import Procedures(进出口贸易的程序)1.5 Overview of This Book (本书的基本内容)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Specimens(单证样本)Chapter Two International Trade Terms(第二章国际贸易术语)2.1 Three Sets of Rules (三种贸易术语的解释规则)2.2 Basics of Incoterms 2010 (2010年国际贸易术语解释通则基本概念)2.3 Application Issues(贸易术语在使用中应注意的问题)2.4 Determinants of Choice of Trade Terms (贸易术语选用的决定因素)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Chapter Three Export Price(第三章出口商品的价格)3.1 Expression of Export Price(出口价格的表达)3.2 Pricing Considerations(影响定价的因素)3.3 Calculation of Price(价格的计算)3.4 Understanding the Price(价格的评估)3.5 Communication of Price(价格的沟通)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Chapter Four Terms of Commodity(第四章商品条款)4.1 Name of Commodity (商品的名称)4.2 Specifying Quality(商品的品质)4.3 Measuring Quantity(商品的数量)4.4 Packing and Marking(商品的包装及标志)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Chapter Five Cargo Transportation(第五章国际货物运输)5.1 Ocean Transportation (海洋运输)5.2 Other Modes of Transportation (其他运输方式)5.3 Transportation Documents(运输单据)5.4 Shipment Clause in the Sales Contract(销售合同中的装运条款)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Specimens(单证样本)Chapter Six Cargo Transportation Insurance(第六章货物运输保险)6.1 Fundamental Principles of Cargo Insurance(货物保险的基本原则)6.2 Marine Risks and Losses(海上风险和损失)6.3 Coverage of Marine Cargo Insurance of CIC(我国海上货物保险范围)6.4 Coverage of Marine Cargo Insurance of ICC(协会货物保险范围)6.5 Other Types of Cargo Insurance(其他货物保险的种类)6.6 Procedures of Cargo Insurance(货物保险程序)6.7 Insurance Terms in the Sales Contract(销售合同中的保险条款)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Specimens(单证样本)Chapter Seven International Payments(第七章国际货款支付)7.1 Issues in Concern(影响支付条件的因素)7.2 Paying Instruments(支付工具)7.3 Remittance(汇付)7.4 Collection(托收)7.5 Basics of Letter of Credit(信用证基础知识)7.6 Types of Documentary Credit(跟单信用证的种类)7.7 Letter of Guarantee(L/G)(保函)7.8 Export Financing(出口融资)7.9 Payment Problems(支付中出现的问题)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Specimens(单证样本)Chapter Eight Export Documentation(第八章出口单证)8.1 Significance of Documentation(单证的重要性)8.2 Basic Requirements for Documentation(单证的基本要求)8.3 Prerequisites of Documentation(制单的依据)8.4 Export Documents(出口单证的种类)8.5 Clause Concerning Documents in the Sales Contract(销售合同中有关单证的条款)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Specimens(单证样本)Chapter Nine Inspection, Claim, Force Majeure and Arbitration(第九章商检、索赔、不可抗力和仲裁)9.1 Commodity Inspection(商品检验)9.2 Disputes and Claims(争议和索赔)9.3 Force Majeure(不可抗力)9.4 Arbitration(仲裁)Summary(总结)Key Terms(主要术语)Abbreviations(缩略语)Exercises(练习)Key to Exercises(练习答案)Glossary(词汇表)Appendix 1INCOTERMS 2010 (FOB, CFR, CIF)(附录12010年国际贸易术语解释通则(FOB,CFR,CIF))Appendix 2CISG 1980 (Part II)(附录2联合国国际货物销售合同公约1980(第二部分)) References (参考书目)帅建林. International Trade Practice. University of International Business and Economics press. 2007.9CONTENTSPart 1 OverviewChapter 1 Introduction to International TradeChapter 2 International Trade PolicyChapter 3 Trade Bloc and Trade BlockChapter 4 WTO :A Navigation GuidePart 2 Terms of International TradeChapter 5 International Trade TermsChapter Terms of CommodityChapter International Cargo TransportChapter 8 Cargo InsuranceChapter 9 Terms of PriceChapter 10 International Payment and SettlementChapter 11 Claims, Force Majeure and ArbitrationPart 3 International Trade ProcedureChapter 12 Launching a Profitable TransactionChapter 13 Business Negotiation and Establishment of ContractChapter 14 Exporting ElementsChapter 15 Importing ElementsChapter 16 DocumentationPart 4 Trade FormsChapter 17 Agency, Distribution and ConsignmentChapter 18 TendersChapter 19 Counter TradeChapter 20 Futures TradingChapter 21 E-CommerceAppendix Glossary of International Trade Terms with English-Chinese InterpretationsBibliographyPaul R.Krugman & Maurice Obstfeld. International Economics:Theory andPolicy,8E. Tsinghua University press. 2011-11Contents前言第1章绪论第1部分国际贸易理论第2章世界贸易:概览第3章劳动生产率和比较优势:李嘉图模型第4章资源、比较优势和收入分配第5章标准贸易模型第6章规模经济、不完全竞争和国际贸易第7章国际要素流动第2部分国际贸易政策第8章贸易政策工具第9章贸易政策中的政治经济学第10章发展中国家的贸易政策第11章贸易政策中的争论数学附录第4章附录要素比例模型第5章附录贸易下的世界经济第6章附录垄断竞争模模型张素芳,International trade: theory and practice. University of International Business & Economics Press, Beijing, 2010contentsSection I. International Trade Theory and PolicyCHAPTER 1.INTRODUCTION TO INTERNATIONAL TRADE1.The Reasons for International Trade1.1. Resources reasons1.2. Economic reasons1.3. Other reasons2. The Differences between International Trade and Domestic Trade'.'2.1. More plex context2.2. More difficult and risky2.3. Higher skills required3.Basic Concepts Relating to International Trade3.1. Visible trade and invisible trade3.2. Favorable balance of trade and unfavorable balance oft rade3.3. General trade system and special trade system3.4. Volume of international trade and quantum of international trade3.5. Commodity position of international trade3.6. Geographical position of international trade3.7. Degree of dependence on foreign tradeCHAPTER 2.CLASSICAL TRADE THEORIES1.Mercantilism1.1. The development of mercantilist thought1.2. The mercantilist economic system1.3. Economic policies pursued by the mercantilists1.4. Discussions2.David Hume's Challenge to Mercantilism2.1. Assumptions of price-specie=flow mechanism2.2. The price-specie-flow mechanism3.Adam Smith's Theory of Absolute Advantage3.1. Assumptions of Adam Smith's theory of absolute advantage3.2. Challenge to Mercantilism3.3. Example4.David Ricardo's Theory of Comparative Advantage4.1. The concept of parative advantage4.2. Example4.3. Analysis of the theory of parative advantage by using modemtools. CHAPTER 3.NEOCLASSICAL TRADE THEORIES.1.Gains from Trade in Neoclassical Trade Theory1.1. Increasing opportunity costs on the PPF1.2. General equilibrium and gains in autarky1.3. General equilibrium and gains after the introduction of international trade ...2.Reciprocal Demand Theory2.1. A country's offer curve2.2. Trading equilibrium2.3. Measurement of terms of trade3.Factor Endowment Theory3.1. Factor intensity in production3.2. Factor endowments, factor prices, and parative advantage3.3. Assumptions of the factor proportions theory.,3.4. The Heckscher-Ohlin theorem.:3.5. An example to illustrate H-O theorem.3.6. The factor price equalization theorem:3.7. The Stolper-Samuelson theorem4.The Leontief Paradox——An Empirical Test of the Factor Proportions Theory 4.1. The Leontief paradox.-4.2. Suggested explanations for the Leontief Paradox and related theories CHAPTER 4.POST-HECKSHER-OHLIN THEORIES OF TRADE1.The Product Cycle Theory1.1. The imitation lag hypothesis1.2. The product cycle theory2.The Linder Theory2.1. Assumptions of the Linder theory2.2. Trade es in the overlapping ranges of products ophistication.:3.Intra-Industry Trade Theory3.1. Explanations of intra-industry trade3.2. Measurement of intra-industry tradeCHAPTER 5.IMPORT PROTECTION POLICY: TARIFFS1.Types of Import Tariffs1.1. In terms of the means of collection1.2. In terms of the different tariff rates applied1.3. In terms of special purposes for collection2.The Effects of Import Tariffs2.1. Concepts of consumer surplus and producer surplus2.2. The welfare effects of import tariffs3.Measurement of Import Tariffs3.1. The 'height' of import tariffs3.2. Nominal versus effective tariff ratesCHAPTER 6.IMPORT PROTECTION POLICY: NON-TARIFF BARRIERS''1.Forms of Non-tariff Barriers.1.1. Quantity control measures1.2. Price control measures1.3. Para-tariff measures1.4. Finance measures1.5. Anti-petitive measures.,.1.6. Miscellaneous measures2.Effects of Non-tariff Barriers2.1. The effects of an import quota2.2. The effects of a subsidy to an import-peting industryCHAPTER 7.EXPORT PROMOTION AND OTHER POLICIES1.Export Subsidy and Production Subsidy1.1. Export subsidy and its effects1.2. Production subsidy and its effects.2.Other Export Promotion Policies2.1. Devaluation of home currency.2.2. Commodity dumping2.3. Bonded warehouse2.4. Special trade zone2.5. Export promotion programs3.Export Restrictions and Import Promotion Policies3.1. Export restrictions policies3.2. Import promotion policies4.Trade Sanctions4.1. Introduction to trade sanctions4.2. Effectiveness of trade sanctionsCHAPTER 8.ARGUMENTS AGAINST FREE TRADE1.Traditional Arguments against Free Trade1.1. Infant industry argument.1.2. Terms of trade argument1.3. Balance of trade argument1.4. Tariff to reduce aggregate unemployment argument1.5. Fair petition argument1.6. National security argument2.New Protectionism2.1. Tariff to extract foreign monopoly profit2.2. Export subsidy in duopoly3.The Political Economy of Trade Policy3.1. Median voter model3.2. Collective action theory.3.3. Contribution in political campaignsCHAPTER 9.REGIONAL ECONOMIC INTEGRATIONof Regional Economic Integration1.1. Preferential tariff arrangement1.2. Free trade area1.3. Customs union1.4. Common market.1.5. Economic union2.The Static and Dynamic Effects of Regional Economic Integration2.1. Static effects of regional economic integration2.2. Dynamic effects of regional economic integration3.Economic Integration in Europe, North America and Asia3.1. Economic integration in Europe……………………………………Chapter 10 International Cargo Transportation InsuranceChapter 11 International Trade PaymentChapter 12 Inspection,Claim,Force Majeure and ArbitrationChapter 13 Trade Negotiation and Formation of the ContractChapter 14 Implementation of the Contract丹尼斯·R·阿普尔亚德 & 小艾尔弗雷德·J·菲尔德 & 史蒂文·L·科布.国际贸易.中国人民大学出版社. 2012-7第1章国际经济学的世界第一部分古典贸易理论第2章早期的国际贸易理论:由重商主义向大卫·李嘉图的古典贸易理论的演进第3章大卫·李嘉图的古典贸易理论和比较优势第4章对古典贸易模型的扩充及验证第二部分新贸易理论第5章新古典贸易理论——基本分析工具的介绍第6章新古典贸易理论中的贸易利得第7章贸易提供曲线和贸易条件第8章贸易的基础:要素禀赋理论和赫克歇尔俄林模型第9章要素禀赋理论的实证分析第三部分贸易理论的扩展第10章后赫克歇尔俄林贸易理论与产业内贸易第11章经济增长与国际贸易第12章国际要素流动第四部分贸易政策第13章贸易政策工具第14章贸易政策的影响第15章对干涉主义贸易政策的争论第16章经济的政治因素与美国的对外贸易政策第17章经济一体化第18章国际贸易与发展中国家参考文献当我被上帝造出来时,上帝问我想在人间当一个怎样的人,我不假思索的说,我要做一个伟大的世人皆知的人。
Reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease trans
Reproduction numbers and sub-threshold endemicequilibria for compartmental models of disease transmissionP.van den Driesschea,1,James Watmough b,*,2aDepartment of Mathematics and Statistics,University of Victoria,Victoria,BC,Canada V8W 3P4b Department of Mathematics and Statistics,University of New Brunswick,Fredericton,NB,Canada E3B 5A3Received 26April 2001;received in revised form 27June 2001;accepted 27June 2001Dedicated to the memory of John JacquezAbstractA precise definition of the basic reproduction number,R 0,is presented for a general compartmental disease transmission model based on a system of ordinary differential equations.It is shown that,if R 0<1,then the disease free equilibrium is locally asymptotically stable;whereas if R 0>1,then it is unstable.Thus,R 0is a threshold parameter for the model.An analysis of the local centre manifold yields a simple criterion for the existence and stability of super-and sub-threshold endemic equilibria for R 0near one.This criterion,together with the definition of R 0,is illustrated by treatment,multigroup,staged progression,multistrain and vector–host models and can be applied to more complex models.The results are significant for disease control.Ó2002Elsevier Science Inc.All rights reserved.Keywords:Basic reproduction number;Sub-threshold equilibrium;Disease transmission model;Disease control1.IntroductionOne of the most important concerns about any infectious disease is its ability to invade a population.Many epidemiological models have a disease free equilibrium (DFE)at whichtheMathematical Biosciences 180(2002)29–48/locate/mbs*Corresponding author.Tel.:+1-5064587323;fax:+1-5064534705.E-mail addresses:pvdd@math.uvic.ca (P.van den Driessche),watmough@unb.ca (J.Watmough).URL:http://www.math.unb.ca/$watmough.1Research supported in part by an NSERC Research Grant,the University of Victoria Committee on faculty research and travel and MITACS.2Research supported by an NSERC Postdoctoral Fellowship tenured at the University of Victoria.0025-5564/02/$-see front matter Ó2002Elsevier Science Inc.All rights reserved.PII:S0025-5564(02)00108-630P.van den Driessche,J.Watmough/Mathematical Biosciences180(2002)29–48population remains in the absence of disease.These models usually have a threshold parameter, known as the basic reproduction number,R0,such that if R0<1,then the DFE is locally as-ymptotically stable,and the disease cannot invade the population,but if R0>1,then the DFE is unstable and invasion is always possible(see the survey paper by Hethcote[1]).Diekmann et al.[2]define R0as the spectral radius of the next generation matrix.We write down in detail a general compartmental disease transmission model suited to heterogeneous populations that can be modelled by a system of ordinary differential equations.We derive an expression for the next generation matrix for this model and examine the threshold R0¼1in detail.The model is suited to a heterogeneous population in which the vital and epidemiological parameters for an individual may depend on such factors as the stage of the disease,spatial position,age or behaviour.However,we assume that the population can be broken into homo-geneous subpopulations,or compartments,such that individuals in a given compartment are indistinguishable from one another.That is,the parameters may vary from compartment to compartment,but are identical for all individuals within a given compartment.We also assume that the parameters do not depend on the length of time an individual has spent in a compart-ment.The model is based on a system of ordinary equations describing the evolution of the number of individuals in each compartment.In addition to showing that R0is a threshold parameter for the local stability of the DFE, we apply centre manifold theory to determine the existence and stability of endemic equilib-ria near the threshold.We show that some models may have unstable endemic equilibria near the DFE for R0<1.This suggests that even though the DFE is locally stable,the disease may persist.The model is developed in Section2.The basic reproduction number is defined and shown to bea threshold parameter in Section3,and the definition is illustrated by several examples in Section4.The analysis of the centre manifold is presented in Section5.The epidemiological ramifications of the results are presented in Section6.2.A general compartmental epidemic model for a heterogeneous populationConsider a heterogeneous population whose individuals are distinguishable by age,behaviour, spatial position and/or stage of disease,but can be grouped into n homogeneous compartments.A general epidemic model for such a population is developed in this section.Let x¼ðx1;...;x nÞt, with each x i P0,be the number of individuals in each compartment.For clarity we sort the compartments so that thefirst m compartments correspond to infected individuals.The distinc-tion between infected and uninfected compartments must be determined from the epidemiological interpretation of the model and cannot be deduced from the structure of the equations alone,as we shall discuss below.It is plausible that more than one interpretation is possible for some models.A simple epidemic model illustrating this is given in Section4.1.The basic reproduction number can not be determined from the structure of the mathematical model alone,but depends on the definition of infected and uninfected compartments.We define X s to be the set of all disease free states.That isX s¼f x P0j x i¼0;i¼1;...;m g:In order to compute R0,it is important to distinguish new infections from all other changes inpopulation.Let F iðxÞbe the rate of appearance of new infections in compartment i,Vþi ðxÞbe therate of transfer of individuals into compartment i by all other means,and VÀi ðxÞbe the rate oftransfer of individuals out of compartment i.It is assumed that each function is continuously differentiable at least twice in each variable.The disease transmission model consists of non-negative initial conditions together with the following system of equations:_x i¼f iðxÞ¼F iðxÞÀV iðxÞ;i¼1;...;n;ð1Þwhere V i¼VÀi ÀVþiand the functions satisfy assumptions(A1)–(A5)described below.Sinceeach function represents a directed transfer of individuals,they are all non-negative.Thus,(A1)if x P0,then F i;Vþi ;VÀiP0for i¼1;...;n.If a compartment is empty,then there can be no transfer of individuals out of the compartment by death,infection,nor any other means.Thus,(A2)if x i¼0then VÀi ¼0.In particular,if x2X s then VÀi¼0for i¼1;...;m.Consider the disease transmission model given by(1)with f iðxÞ,i¼1;...;n,satisfying con-ditions(A1)and(A2).If x i¼0,then f iðxÞP0and hence,the non-negative cone(x i P0, i¼1;...;n)is forward invariant.By Theorems1.1.8and1.1.9of Wiggins[3,p.37]for each non-negative initial condition there is a unique,non-negative solution.The next condition arises from the simple fact that the incidence of infection for uninfected compartments is zero.(A3)F i¼0if i>m.To ensure that the disease free subspace is invariant,we assume that if the population is free of disease then the population will remain free of disease.That is,there is no(density independent) immigration of infectives.This condition is stated as follows:(A4)if x2X s then F iðxÞ¼0and VþiðxÞ¼0for i¼1;...;m.The remaining condition is based on the derivatives of f near a DFE.For our purposes,we define a DFE of(1)to be a(locally asymptotically)stable equilibrium solution of the disease free model,i.e.,(1)restricted to X s.Note that we need not assume that the model has a unique DFE. Consider a population near the DFE x0.If the population remains near the DFE(i.e.,if the introduction of a few infective individuals does not result in an epidemic)then the population will return to the DFE according to the linearized system_x¼Dfðx0ÞðxÀx0Þ;ð2Þwhere Dfðx0Þis the derivative½o f i=o x j evaluated at the DFE,x0(i.e.,the Jacobian matrix).Here, and in what follows,some derivatives are one sided,since x0is on the domain boundary.We restrict our attention to systems in which the DFE is stable in the absence of new infection.That is, (A5)If FðxÞis set to zero,then all eigenvalues of Dfðx0Þhave negative real parts.P.van den Driessche,J.Watmough/Mathematical Biosciences180(2002)29–4831The conditions listed above allow us to partition the matrix Df ðx 0Þas shown by the following lemma.Lemma 1.If x 0is a DFE of (1)and f i ðx Þsatisfies (A1)–(A5),then the derivatives D F ðx 0Þand D V ðx 0Þare partitioned asD F ðx 0Þ¼F 000 ;D V ðx 0Þ¼V 0J 3J 4;where F and V are the m Âm matrices defined byF ¼o F i o x j ðx 0Þ !and V ¼o V i o x jðx 0Þ !with 16i ;j 6m :Further ,F is non-negative ,V is a non-singular M-matrix and all eigenvalues of J 4have positive real part .Proof.Let x 02X s be a DFE.By (A3)and (A4),ðo F i =o x j Þðx 0Þ¼0if either i >m or j >m .Similarly,by (A2)and (A4),if x 2X s then V i ðx Þ¼0for i 6m .Hence,ðo V i =o x j Þðx 0Þ¼0for i 6m and j >m .This shows the stated partition and zero blocks.The non-negativity of F follows from (A1)and (A4).Let f e j g be the Euclidean basis vectors.That is,e j is the j th column of the n Ân identity matrix.Then,for j ¼1;...;m ,o V i o x jðx 0Þ¼lim h !0þV i ðx 0þhe j ÞÀV i ðx 0Þh :To show that V is a non-singular M-matrix,note that if x 0is a DFE,then by (A2)and (A4),V i ðx 0Þ¼0for i ¼1;...;m ,and if i ¼j ,then the i th component of x 0þhe j ¼0and V i ðx 0þhe j Þ60,by (A1)and (A2).Hence,o V i =o x j 0for i m and j ¼i and V has the Z sign pattern (see Appendix A).Additionally,by (A5),all eigenvalues of V have positive real parts.These two conditions imply that V is a non-singular M-matrix [4,p.135(G 20)].Condition (A5)also implies that the eigenvalues of J 4have positive real part.Ã3.The basic reproduction numberThe basic reproduction number,denoted R 0,is ‘the expected number of secondary cases produced,in a completely susceptible population,by a typical infective individual’[2];see also [5,p.17].If R 0<1,then on average an infected individual produces less than one new infected individual over the course of its infectious period,and the infection cannot grow.Conversely,if R 0>1,then each infected individual produces,on average,more than one new infection,and the disease can invade the population.For the case of a single infected compartment,R 0is simply the product of the infection rate and the mean duration of the infection.However,for more complicated models with several infected compartments this simple heuristic definition of R 0is32P.van den Driessche,J.Watmough /Mathematical Biosciences 180(2002)29–48insufficient.A more general basic reproduction number can be defined as the number of new infections produced by a typical infective individual in a population at a DFE.To determine the fate of a‘typical’infective individual introduced into the population,we consider the dynamics of the linearized system(2)with reinfection turned off.That is,the system _x¼ÀD Vðx0ÞðxÀx0Þ:ð3ÞBy(A5),the DFE is locally asymptotically stable in this system.Thus,(3)can be used to de-termine the fate of a small number of infected individuals introduced to a disease free population.Let wi ð0Þbe the number of infected individuals initially in compartment i and letwðtÞ¼w1ðtÞ;...;w mðtÞðÞt be the number of these initially infected individuals remaining in the infected compartments after t time units.That is the vector w is thefirst m components of x.The partitioning of D Vðx0Þimplies that wðtÞsatisfies w0ðtÞ¼ÀV wðtÞ,which has the unique solution wðtÞ¼eÀVt wð0Þ.By Lemma1,V is a non-singular M-matrix and is,therefore,invertible and all of its eigenvalues have positive real parts.Thus,integrating F wðtÞfrom zero to infinity gives the expected number of new infections produced by the initially infected individuals as the vector FVÀ1wð0Þ.Since F is non-negative and V is a non-singular M-matrix,VÀ1is non-negative[4,p.137 (N38)],as is FVÀ1.To interpret the entries of FVÀ1and develop a meaningful definition of R0,consider the fate of an infected individual introduced into compartment k of a disease free population.The(j;k)entry of VÀ1is the average length of time this individual spends in compartment j during its lifetime, assuming that the population remains near the DFE and barring reinfection.The(i;j)entry of F is the rate at which infected individuals in compartment j produce new infections in compartment i. Hence,the(i;k)entry of the product FVÀ1is the expected number of new infections in com-partment i produced by the infected individual originally introduced into compartment k.Fol-lowing Diekmann et al.[2],we call FVÀ1the next generation matrix for the model and set R0¼qðFVÀ1Þ;ð4Þwhere qðAÞdenotes the spectral radius of a matrix A.The DFE,x0,is locally asymptotically stable if all the eigenvalues of the matrix Dfðx0Þhave negative real parts and unstable if any eigenvalue of Dfðx0Þhas a positive real part.By Lemma1, the eigenvalues of Dfðx0Þcan be partitioned into two sets corresponding to the infected and uninfected compartments.These two sets are the eigenvalues of FÀV and those ofÀJ4.Again by Lemma1,the eigenvalues ofÀJ4all have negative real part,thus the stability of the DFE is determined by the eigenvalues of FÀV.The following theorem states that R0is a threshold parameter for the stability of the DFE.Theorem2.Consider the disease transmission model given by(1)with fðxÞsatisfying conditions (A1)–(A5).If x0is a DFE of the model,then x0is locally asymptotically stable if R0<1,but un-stable if R0>1,where R0is defined by(4).Proof.Let J1¼FÀV.Since V is a non-singular M-matrix and F is non-negative,ÀJ1¼VÀF has the Z sign pattern(see Appendix A).Thus,sðJ1Þ<0()ÀJ1is a non-singular M-matrix;P.van den Driessche,J.Watmough/Mathematical Biosciences180(2002)29–483334P.van den Driessche,J.Watmough/Mathematical Biosciences180(2002)29–48where sðJ1Þdenotes the maximum real part of all the eigenvalues of the matrix J1(the spectral abscissa of J1).Since FVÀ1is non-negative,ÀJ1VÀ1¼IÀFVÀ1also has the Z sign pattern.Ap-plying Lemma5of Appendix A,with H¼V and B¼ÀJ1¼VÀF,we have ÀJ1is a non-singular M-matrix()IÀFVÀ1is a non-singular M-matrix:Finally,since FVÀ1is non-negative,all eigenvalues of FVÀ1have magnitude less than or equal to qðFVÀ1Þ.Thus,IÀFVÀ1is a non-singular M-matrix;()qðFVÀ1Þ<1:Hence,sðJ1Þ<0if and only if R0<1.Similarly,it follows thatsðJ1Þ¼0()ÀJ1is a singular M-matrix;()IÀFVÀ1is a singular M-matrix;()qðFVÀ1Þ¼1:The second equivalence follows from Lemma6of Appendix A,with H¼V and K¼F.The remainder of the equivalences follow as with the non-singular case.Hence,sðJ1Þ¼0if and only if R0¼1.It follows that sðJ1Þ>0if and only if R0>1.ÃA similar result can be found in the recent book by Diekmann and Heesterbeek[6,Theorem6.13].This result is known for the special case in which J1is irreducible and V is a positive di-agonal matrix[7–10].The special case in which V has positive diagonal and negative subdiagonal elements is proven in Hyman et al.[11,Appendix B];however,our approach is much simpler(see Section4.3).4.Examples4.1.Treatment modelThe decomposition of fðxÞinto the components F and V is illustrated using a simple treat-ment model.The model is based on the tuberculosis model of Castillo-Chavez and Feng[12,Eq.(1.1)],but also includes treatment failure used in their more elaborate two-strain model[12,Eq.(2.1)].A similar tuberculosis model with two treated compartments is proposed by Blower et al.[13].The population is divided into four compartments,namely,individuals susceptible to tu-berculosis(S),exposed individuals(E),infectious individuals(I)and treated individuals(T).The dynamics are illustrated in Fig.1.Susceptible and treated individuals enter the exposed com-partment at rates b1I=N and b2I=N,respectively,where N¼EþIþSþT.Exposed individuals progress to the infectious compartment at the rate m.All newborns are susceptible,and all indi-viduals die at the rate d>0.Thus,the core of the model is an SEI model using standard inci-dence.The treatment rates are r1for exposed individuals and r2for infectious individuals. However,only a fraction q of the treatments of infectious individuals are successful.Unsuc-cessfully treated infectious individuals re-enter the exposed compartment(p¼1Àq).The diseasetransmission model consists of the following differential equations together with non-negative initial conditions:_E¼b1SI=Nþb2TI=NÀðdþmþr1ÞEþpr2I;ð5aÞ_I¼m EÀðdþr2ÞI;ð5bÞ_S¼bðNÞÀdSÀb1SI=N;ð5cÞ_T¼ÀdTþr1Eþqr2IÀb2TI=N:ð5dÞProgression from E to I and failure of treatment are not considered to be new infections,but rather the progression of an infected individual through the various compartments.Hence,F¼b1SI=Nþb2TI=NB B@1C CA and V¼ðdþmþr1ÞEÀpr2IÀm Eþðdþr2ÞIÀbðNÞþdSþb1SI=NdTÀr1EÀqr2Iþb2TI=NB B@1C CA:ð6ÞThe infected compartments are E and I,giving m¼2.An equilibrium solution with E¼I¼0has the form x0¼ð0;0;S0;0Þt,where S0is any positive solution of bðS0Þ¼dS0.This will be a DFE if and only if b0ðS0Þ<d.Without loss of generality,assume S0¼1is a DFE.Then,F¼0b100;V¼dþmþr1Àpr2Àm dþr2;givingVÀ1¼1ðdþmþr1Þðdþr2ÞÀm pr2dþr2pr2m dþmþr1and R0¼b1m=ððdþmþr1Þðdþr2ÞÀm pr2Þ.A heuristic derivation of the(2;1)entry of VÀ1and R0are as follows:a fraction h1¼m=ðdþmþr1Þof exposed individuals progress to compartment I,a fraction h2¼pr2=ðdþr2Þof infectious individuals re-enter compartment E.Hence,a fractionh1of exposed individuals pass through compartment I at least once,a fraction h21h2passthroughat least twice,and a fraction h k 1h k À12pass through at least k times,spending an average of s ¼1=ðd þr 2Þtime units in compartment I on each pass.Thus,an individual introduced into com-partment E spends,on average,s ðh 1þh 21h 2þÁÁÁÞ¼s h 1=ð1Àh 1h 2Þ¼m =ððd þm þr 1Þðd þr 2ÞÀm pr 2Þtime units in compartment I over its expected lifetime.Multiplying this by b 1gives R 0.The model without treatment (r 1¼r 2¼0)is an SEI model with R 0¼b 1m =ðd ðd þm ÞÞ.The interpretation of R 0for this case is simpler.Only a fraction m =ðd þm Þof exposed individuals progress from compartment E to compartment I ,and individuals entering compartment I spend,on average,1=d time units there.Although conditions (A1)–(A5)do not restrict the decomposition of f i ðx Þto a single choice for F i ,only one such choice is epidemiologically correct.Different choices for the function F lead to different values for the spectral radius of FV À1,as shown in Table 1.In column (a),treatment failure is considered to be a new infection and in column (b),both treatment failure and pro-gression to infectiousness are considered new infections.In each case the condition q ðFV À1Þ<1yields the same portion of parameter space.Thus,q ðFV À1Þis a threshold parameter in both cases.The difference between the numbers lies in the epidemiological interpretation rather than the mathematical analysis.For example,in column (a),the infection rate is b 1þpr 2and an exposed individual is expected to spend m =ððd þm þr 1Þðd þr 2ÞÞtime units in compartment I .However,this reasoning is biologically flawed since treatment failure does not give rise to a newly infected individual.Table 1Decomposition of f leading to alternative thresholds(a)(b)Fb 1SI =N þb 2TI =N þpr 2I 0000B B @1C C A b 1SI =N þb 2TI =N þpr 2I m E 000B B @1C C A Vðd þm þr 1ÞE Àm E þðd þr 2ÞI Àb ðN ÞþdS þb 1SI =N dT Àr 1E Àqr 2I þb 2TI =N 0B B @1C C A ðd þm þr 1ÞE ðd þr 2ÞI Àb ðN ÞþdS þb 1SI =N dT Àr 1E Àqr 2I þb 2TI =N 0B B @1C C A F0b 1þpr 200 0b 1þpr 2m 0 V d þm þr 10Àm d þr 2d þm þr 100d þr 2 q (FV À1)b 1m þpr 2mðd þm þr 1Þðd þr 2Þffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffib 1m þpr 2mðd þm þr 1Þðd þr 2Þs 36P.van den Driessche,J.Watmough /Mathematical Biosciences 180(2002)29–484.2.Multigroup modelIn the epidemiological literature,the term‘multigroup’usually refers to the division of a het-erogeneous population into several homogeneous groups based on individual behaviour(e.g., [14]).Each group is then subdivided into epidemiological compartments.The majority of mul-tigroup models in the literature are used for sexually transmitted diseases,such as HIV/AIDS or gonorrhea,where behaviour is an important factor in the probability of contracting the disease [7,8,14,15].As an example,we use an m-group SIRS-vaccination model of Hethcote[7,14]with a generalized incidence term.The sample model includes several SI multigroup models of HIV/ AIDS as special cases[8,15].The model equations are as follows:_I i ¼X mj¼1b ijðxÞS i I jÀðd iþc iþ iÞI i;ð7aÞ_S i ¼ð1Àp iÞb iÀðd iþh iÞS iþr i R iÀX mj¼1b ijðxÞS i I j;ð7bÞ_Ri¼p i b iþc i I iþh i S iÀðd iþr iÞR i;ð7cÞfor i¼1;...;m,where x¼ðI1;...;I m;S1;...;S m;R1;...;R mÞt.Susceptible and removed individu-als die at the rate d i>0,whereas infected individuals die at the faster rate d iþ i.Infected in-dividuals recover with temporary immunity from re-infection at the rate c i,and immunity lasts an expected1=r i time units.All newborns are susceptible,and a constant fraction b i are born into each group.A fraction p i of newborns are vaccinated at birth.Thereafter,susceptible individuals are vaccinated at the rate h i.The incidence,b ijðxÞdepends on individual behaviour,which determines the amount of mixing between the different groups(see,e.g.,Jacquez et al.[16]). The DFE for this model isx0¼ð0;...;0;S01;...;S0m;R01;...;R0mÞt;whereS0 i ¼b i d ið1Àp iÞþr iðÞd iðd iþh iþr iÞ;R0 i ¼b iðh iþd i p iÞd iðd iþh iþr iÞ:Linearizing(7a)about x¼x0givesF¼S0i b ijðx0ÞÂÃandV¼½ðd iþc iþ iÞd ij ;where d ij is one if i¼j,but zero otherwise.Thus,FVÀ1¼S0i b ijðx0Þ=ðd iÂþc iþ iÞÃ:P.van den Driessche,J.Watmough/Mathematical Biosciences180(2002)29–4837For the special case with b ij separable,that is,b ijðxÞ¼a iðxÞk jðxÞ,F has rank one,and the basic reproduction number isR0¼X mi¼1S0ia iðx0Þk iðx0Þd iþc iþ i:ð8ÞThat is,the basic reproduction number of the disease is the sum of the‘reproduction numbers’for each group.4.3.Staged progression modelThe staged progression model[11,Section3and Appendix B]has a single uninfected com-partment,and infected individuals progress through several stages of the disease with changing infectivity.The model is applicable to many diseases,particularly HIV/AIDS,where transmission probabilities vary as the viral load in an infected individual changes.The model equations are as follows(see Fig.2):_I 1¼X mÀ1k¼1b k SI k=NÀðm1þd1ÞI1;ð9aÞ_Ii¼m iÀ1I iÀ1Àðm iþd iÞI i;i¼2;...;mÀ1;ð9bÞ_Im¼m mÀ1I mÀ1Àd m I m;ð9cÞ_S¼bÀbSÀX mÀ1k¼1b k SI k=N:ð9dÞThe model assumes standard incidence,death rates d i>0in each infectious stage,and thefinal stage has a zero infectivity due to morbidity.Infected individuals spend,on average,1=m i time units in stage i.The unique DFE has I i¼0,i¼1;...;m and S¼1.For simplicity,define m m¼0. Then F¼½F ij and V¼½V ij ,whereF ij¼b j i¼1;j6mÀ1;0otherwise;&ð10ÞV ij¼m iþd i j¼i;Àm j i¼1þj;0otherwise:8<:ð11ÞLet a ij be the(i;j)entry of VÀ1.Thena ij¼0i<j;1=ðm iþd iÞi¼j;Q iÀ1k¼jm kQ ik¼jðm kþd kÞj<i:8>>><>>>:ð12ÞThus,R0¼b1m1þd1þb2m1ðm1þd1Þðm2þd2Þþb3m1m2ðm1þd1Þðm2þd2Þðm3þd3ÞþÁÁÁþb mÀ1m1...m mÀ2ðm1þd1Þ...ðm mÀ1þd mÀ1Þ:ð13ÞThe i th term in R0represents the number of new infections produced by a typical individual during the time it spends in the i th infectious stage.More specifically,m iÀ1=ðm iÀ1þd iÀ1Þis the fraction of individuals reaching stage iÀ1that progress to stage i,and1=ðm iþd iÞis the average time an individual entering stage i spends in stage i.Hence,the i th term in R0is the product of the infectivity of individuals in stage i,the fraction of initially infected individuals surviving at least to stage i,and the average infectious period of an individual in stage i.4.4.Multistrain modelThe recent emergence of resistant viral and bacterial strains,and the effect of treatment on their proliferation is becoming increasingly important[12,13].One framework for studying such sys-tems is the multistrain model shown in Fig.3,which is a caricature of the more detailed treatment model of Castillo-Chavez and Feng[12,Section2]for tuberculosis and the coupled two-strain vector–host model of Feng and Velasco-Hern a ndez[17]for Dengue fever.The model has only a single susceptible compartment,but has two infectious compartments corresponding to the two infectious agents.Each strain is modelled as a simple SIS system.However,strain one may ‘super-infect’an individual infected with strain two,giving rise to a new infection incompartment。
Involvement of Extracellular Signal-Regulated Kinase (ERK) in Pardaxin-Induced Dopamine Rel
Involvement of Extracellular Signal-Regulated Kinase (ERK)in Pardaxin-Induced Dopamine Release from PC12CellsEUGENIA BLOCH-SHILDERMAN,HAO JIANG,SALEH ABU-RAYA,MICHAL LINIAL,1and PHILIP LAZAROVICI 2Department of Pharmacology and Experimental Therapeutics,School of Pharmacy,Faculty of Medicine (E.B.-S.,S.A.-R.,P.L.),andDepartment of Biological Chemistry,Life Sciences Institute (M.L.),The Hebrew University of Jerusalem,Jerusalem,Israel;William T.Gossett Neurology Laboratories,Henry Ford Health Sciences Center,and John D.Dingell Veterans Affairs Medical Center,Detroit,Michigan (H.J.)Received July 13,2000;accepted November 11,2000This paper is available online at ABSTRACTPardaxin (PX),an ionophore-peptide neurotoxin isolated from the fish Pardachirus marmoratus ,induces neurotransmitter re-lease from neuronal preparations by both calcium-dependent and calcium-independent mechanisms.The aim of the present study was to investigate the role of extracellular signal-regu-lated kinase (ERK)/mitogen-activated protein kinase (MAPK)in pardaxin-induced dopamine (DA)release.The experiments were performed on variants of the PC12cell line,an established cellular model for investigating DA release.Time course exper-iments indicated that PX,at nontoxic concentrations,stimu-lated ERK1and ERK2within 5to 15min,measured with a dual phospho-ERK antibody.PX stimulation of ERK activity wascalcium (Ca 2ϩ)-dependent and followed by ERK translocation to the nucleus.This effect was temporally related to PX-in-duced exocytosis,and measured by [3H]dopamine release as well as by a vesicle fusion-based enzyme-linked immunosor-bent assay.Blocking ERK activity with the specific mitogen-activated protein kinase kinase inhibitors PD98059(50M for 45min)and UO126(30M for 30min)inhibited PX-induced exocytosis in the presence but not in the absence of extracel-lular Ca 2ϩ.These results suggest the essential role of ERKs in PX-induced DA release under physiological conditions and support the hypothesis that ERKs are involved in regulating exocytosis.Pardaxin is an amphipathic polypeptide neurotoxin com-posed of 33amino acids,which was isolated from the Red Sea sole,Pardachirus marmoratus (Lazarovici et al.,1986).PX belongs to a family of five PX isoforms isolated from Par-dachirus species living in the Pacific Ocean and the Red Sea (Adermann et al.,1998).In the present study we used mainly Asp-31-PX (P5)due to its lower cytotoxicity compared with that of Gly-31-PX (P4)(Adermann et al.,1998).Pardaxins,which have been shown to form voltage-dependent pores in liposomes (Loew et al.,1985;Lazarovici et al.,1986)and artificial lipid membranes (Lazarovici et al.,1992;Shi et al.,1995),act as polypeptide ionophores.They are considered unique pharmacological tools for studying neurotransmitter release (Lazarovici,1994;Bloch-Shilderman et al.,1997),based on their ability to stimulate exocytosis in different neuronal systems,including brain slices (Wang and Fried-man,1986),neuromuscular junction (Renner et al.,1987),neurosecretory chromaffin cells (Lazarovici and Lelkes,1992),and synaptosomes (Arribas et al.,1993),by both Ca 2ϩ-dependent and Ca 2ϩ-independent mechanisms (Lazarovici and Lelkes,1992;Abu-Raya et al.,1999).PX is thought to act by insertion into the neuronal plasma membrane,leading to the opening of poorly selective cation channels,culminating in depolarization,Ca 2ϩentry,and neurotransmitter release (Lazarovici and Lelkes,1992;Abu-Raya et al.,1999).In an attempt to elucidate the signal transduction pathways in-volved in PX-induced DA release we recently showed that PX stimulates the arachidonic acid cascade in a Ca 2ϩ-dependent and Ca 2ϩ-independent manner in PC12cells (Abu-Raya et al.,1998).A direct relationship was also proposed between PX-stimulation of DA release and the arachidonic acid cas-cade (Abu-Raya et al.,1999).An additional signal transduction circuit that may contrib-ute to PX-induced DA release is the Ras-mitogen-activated protein kinase (MAPK)pathway (Blenis,1993).MAPKs/ERKs are a family of protein-serine/threonine kinases with a diverse array of cellular targets,suggesting that they are key regulators of many cellular responses.They are activated by a wide variety of extracellular stimuli affecting eukaryotic cells,including hormones,growth factors,radicals,mitogens,1M.L.is supported by the Ministry of Health and is a member of the Otto Levi Center for Molecular and Cellular Neurobiology.2P.L.is affiliated with and supported by the David R.Bloom Center for Pharmacy at the Hebrew University.ABBREVIATIONS:PX,pardaxin;DA,dopamine;MAPK,mitogen-activated protein kinase;ERK,extracellular signal-regulated kinase;MEK,MAPK/ERK kinase;MBP,myelin basic protein;NGF,nerve growth factor;DMEM,Dulbecco’s modified Eagle’s medium;PC12,pheochromocy-toma cell line;M-M17-26,a dominant-negative Ras PC12variant cell line;ELISA,enzyme-linked immunosorbent assay;PBS,phosphate-buffered saline;PAGE,polyacrylamide gel electrophoresis;CON,control;WT,wild type.0022-3565/01/2963-704–711$3.00T HE J OURNAL OF P HARMACOLOGY AND E XPERIMENTAL T HERAPEUTICSVol.296,No.3Copyright ©2001by The American Society for Pharmacology and Experimental Therapeutics 3121/885007JPET 296:704–711,2001Printed in U.S.A.704UV,and toxins (Force and Bonventre,1998).These kinases require phosphorylation of both the serine/threonine and ty-rosine residues in the catalytic domain for activation and to be translocated into the nucleus (Treisman,1996).The ERKs are activated by an upstream activator kinase,MEK (MAPK kinase)(Blenis,1993),which is selectively blocked by PD98059and UO126inhibitors (Alessi et al.,1995;Favata et al.,1998).The potential role of ERKs in secretagogue-in-duced norepinephrine release from bovine adrenal chromaf-fin cells was recently proposed (Cox et al.,1996;Cox and Parsons,1997).However,the basic regulatory role of ERKs and their substrates involved in neurotransmitter release have not been fully characterized.The aim of the present study was to assess the role of ERKs in regulating PX-induced DA release from PC12cells.Our results indicate that PX stimulates Ca 2ϩ-dependent ERK activity in PC12cells.This process is essential to PX-induced DA release in Ca 2ϩ-containing medium.Our findings sup-port the importance of ERKs in neurotransmitter release.Experimental ProceduresMaterials[3H]Dopamine (47Ci/mmol),45Ca 2ϩ(5–50mCi/mg),and [␥-32P]ATP were purchased from Amersham,Arlington Heights,IL;HEPES,my-elin basic protein (MBP),trypan blue,sorbitol,bovine serum albumin,EGTA,MgCl 2,CaCl 2,NaCl,NaOH,Triton X-100,glycerol,dithiothre-itol,aprotinin,leupeptin,rabbit protein kinase inhibitor,sodium van-adate,phenylmethylsulfonyl fluoride,-mercaptoethanol,bromphenol blue,ATP,SDS,Nonidet P-40,choline chloride,anti-synaptotagmin antibody,and poly(L -lysine)were purchased from Sigma Chemical Co.,St.Louis,MO;methanol,KCl,and ascorbic acid were purchased from Merck,Darmstadt,Germany;PD98059was purchased from BIOMOL Research Laboratories,Inc.,Plymouth Meeting,PA;UO126was pur-chased from Promega,Madison,WI;anti-phospho ERK and anti-pan ERK antibodies were a generous gift from Dr.Erik Schaefer,QCB,Hopkinton,MA;and nerve growth factor (NGF)was kindly provided by Alomone Labs,Jerusalem,Israel.DMEM,horse and calf serum,anti-biotics,normal goat serum,and rat tail type 1collagen were purchased from Beit Ha’emek,Afula,Israel.The ELISA tetramethylbenzidine developing kit was purchased from Clark Laboratories,Inc.,James-town,NY,and the ELISA blocking reagent was purchased from Sorin Biomedica,Saluggia,Italy;horseradish peroxidase-goat anti-rabbit was purchased from Jackson Immune Research Laboratories,Inc.,West Grove,PA.Protein A agarose was purchased from Amersham Pharma-cia Biotech,Buckinghamshire,UK;and polyethyleneimine was synthe-sized and kindly provided by Prof.A.Domb,Department of Medicinal Chemistry,School of Pharmacy,Hebrew University of Jerusalem,Jerusalem,Israel.ToxinsNative PX (P4)was purified by liquid chromatography from the lyophilized secretion of the flatfish Pardachirus marmoratus (collect-ed in Eilat,Israel)(Lazarovici et al.,1986).Synthetic pardaxins P4and P5were prepared on a 433A peptide synthesizer (PerkinElmer-ABI)using standard Fmoc solid phase chemistry,as previously de-scribed (Adermann et al.,1998).The toxins were purified by high pressure liquid chromatography,using Vydac C18column and ana-lyzed by electrospray mass spectrometry (Adermann et al.,1998).Synthetic pardaxins were kindly provided by Knut Adermann,In-stitute for Peptide Research-PharmaCeuticals GmbH,Hannover,Germany.Cell ViabilityCytotoxicity was determined by trypan blue exclusion (Abu-Raya et al.,1999).The concentrations of PX were considered subcytotoxic when cell death was Ͻ10%.PC12CulturesPC12cells were grown in DMEM supplemented with 7%fetal calf serum,7%horse serum,100g/ml streptomycin,and 100units/ml penicillin.The cultures were maintained in an incubator at 37°C in an atmosphere of 6%CO 2.The medium was changed twice weekly and the cultures were split at a 1:6ratio once a week.In all exper-iments,unless specified otherwise,the cells were plated on six-well dishes coated with equal parts of collagen (0.01mg/ml collagen in 0.1M acetic acid),poly(L -lysine)(0.01mg/ml),and polyethyleneimine (0.1mg/ml).The PC12cell variants M-M17-26,expressing the dom-inant-negative mutant Ha-ras Asn-17gene (Lazarovici et al.,1997),were grown in the presence of 200g/ml G418(Life Technologies,Gaithersburg,MD).[3H]Dopamine ReleaseDA release from PC12cells was determined with slight modifica-tions,as previously described (Abu-Raya et al.,1999).Briefly,fresh DMEM was added and the cells were allowed to equilibrate at 37°C for 30min.The cells were then loaded with [3H]dopamine (0.3–1Ci/ml)for 12h at 37°C.The medium was removed and the cells were washed once with serum-supplemented medium and twice with serum-free medium containing 1mM ascorbic acid.Fresh medium was added,and the cultures were incubated with PX (4M)or KCl (80mM)in the presence (1.8mM)or absence of Ca 2ϩ(ϩ1mM EGTA)for 20min.Basal release was measured in cultures incubated for similar intervals at 37°C and left untreated.Samples of 0.2ml were removed from the medium,centrifuged for 10min (1000g )to remove floating cells,and the radioactivity was measured.To measure total radioactivity,the cells were washed with PBS and dissolved in 1ml of 0.5N NaOH and 0.2-ml aliquots were measured for radioactivity.The data are presented as [3H]dopamine release,calculated as per-centage of control.Vesicle Fusion-Based ELISAThe protocol followed was essentially as previously described (Par-nas and Linial,1998).Briefly,PC12cells were grown on 48-well plates.Control (untreated)cells or cells pretreated with PD98059for 45min were further exposed to PX or PBS for an additional 20min in the presence of polyclonal anti-synaptotagmin I antibody,which was raised against the N-terminal luminal 19amino acids of synap-totagmin I.This soluble N -ethylmaleimide-sensitive fusion factor attachment protein receptor (SNARE)is briefly exposed on the cell surface during the exocytotic process (Parnas and Linial,1998),enabling detection with the antibody.We optimized the antibody concentration to 6g/ml.Upon termination of incubation,the cells were washed three times with PBS at 37°C,with an interval of 15min between washes,and then fixed with 4%paraformaldehyde in PBS for an additional 20min at room temperature.The cells were washed once with 0.5ml of PBS and twice with 0.5ml of PBS containing 0.1%Triton X-100,and incubated with 3%H 2O 2in PBS for 5min.The cells were then incubated for 30min at 25°C with blocking solution (5%normal goat serum,2%bovine serum albumin,0.1%Triton X-100in PBS)and then with goat anti-rabbit antibody conjugated to peroxidase (0.35ml,diluted 1:7500)for 1h at 25°C.Cells were further washed three times with PBS containing 0.1%Triton X-100.After washing,the cells were incubated with 1:1chromagen and substrate solution (150l)for 5min.Every 10s,75-l samples from each well were removed and mixed with the stop solution (150l,1N sulfuric acid).The intensity of the blue color formed was monitored at OD 450nm with the aid of an ELISA reader-Kinetic analyzer V-MAX with the attached software SOFTmax (Mo-lecular Devices Corporation,Menlo Park,CA).Control experimentsInvolvement of ERK in Pardaxin-Induced Dopamine Release705with secondary antibody alone were used to subtract the background value.Staining with nonrelevant antibodies was included routinely. Total immunoreactivity was measured by fixing the cells and incu-bating with synaptotagmin I antibodies in blocking solution(0.2g/ml),followed by incubation with secondary antibody as described above.Calcium UptakeThe procedure is described in detail in Jiang et al.(1997).Briefly, cells were grown in six-well plates in DMEM containing10%fetal bovine serum.After24h at37°C,cells were washed once with Ca2ϩand serum/antibiotic-free medium and incubated in the same me-dium for30min.45Ca2ϩ(1Ci)was then added to each well along with PX(4M)or KCl(80mM)and the cells were incubated at37°C for an additional4min.The medium was rapidly aspirated,and the cells were washed twice with3ml of wash buffer,pH7.2,containing 20mM HEPES,50mM choline chloride,and2.5mM EGTA.The cells were lysed in0.5ml of NaOH for2h.A0.4-ml sample of the cell lysate was used to estimate the cell-associated radioactivity and0.05 ml was used for protein determination.The final counts were nor-malized per milligram of protein(cpm/mg of protein)and the data are presented as percentage of control.ERK AssaysDetection of Activated ERKs in PC12Cell Extracts by West-ern Blotting Using Anti-Phospho ERK Polyclonal Antibodies. Activation of MAPKs/ERKs requires that these enzymes are dually phosphorylated by MEK(Blenis,1993)on both the Thr and the Tyr residues in the Thr-Glu-Tyr consensus sequence within the catalytic core of the enzyme(Payne et al.,1991).The phospho-ERK antibody (anti-active ERK)used in the present study was developed against a dually phosphorylated synthetic peptide encompassing residues Thr-183and Tyr-185of p42/MAPK2/erk2,corresponding to the active form of the ERK enzymes(Khokhlatchev et al.,1997).The antibody was purified,using a negative adsorption step to remove antibody recognizing the nonphosphopeptide,followed by positive selection-affinity purification with the dually phosphorylated peptide to select for antibody preferentially recognizing ERK1and ERK2.PC12cells were treated with50ng/ml NGF for5min,or with0.5M sorbitol for 5min or PX for15min,unless otherwise indicated,or left untreated. The cells were lysed and aliquots(50g)of each extract were ana-lyzed by SDS-PAGE(10%gel,under reducing conditions)and trans-ferred to a nitrocellulose membrane.The membranes were probed with the indicated anti-phospho antibody,which recognizes the ac-tive(phosphorylated)forms of ERKs or with anti-pan ERK antibody, a polyclonal antibody that recognizes the inactive(nonphosphory-lated)form of ERKs,at a dilution of1:10,000(Lazarovici et al.,1998).Measurement of ERK Activity,Using MBP as Substrate.In another approach we measured ERK activity by protein phosphory-lation assay,using the substrate MBP.MBP serves as a general nonselective substrate for a variety of protein kinases(Chan and Lazarovici,1987).The first step was the immunoprecipitation of ERKs from the cell lysates,using anti-ERK polyclonal antibody(at a dilution of1:200for2-h incubation at37°C with continuous agita-tion).Thereafter,triplicate lysates of the various samples were in-cubated with washed protein A agarose(50-l suspension)for addi-tional2-h incubation at4°C.The immunoprecipitated ERK,bound to protein A agarose was washed twice with0.5ml of kinase assay buffer.In the second step,the washed immunoprecipitated ERK, bound to protein A agarose,was suspended in30l of kinase assay buffer{7.5mM HEPES,pH7.5,10mM MgCl2,1mM dithiothreitol, 2.5M protein kinase A inhibitor peptide PKI(6-22)-amide,225M cold ATP,25Ci of[␥-32P]ATP,and500g/ml MBP},and incubated for30min at room temperature.The reaction was terminated by the addition of30l of SDS sample buffer,heated for5min and elec-trophoresed on SDS-PAGE.Phosphorylated MBP was visualized by autoradiography(XAR film;Eastman Kodak,Rochester,NY).The bands were quantified with a laser scanner.Immunoblotting was performed as previously described(Laz-arovici et al.,1998).Briefly,cells were plated on10-cm Petri dishes 24h before the experiment.After treatment with PX or other re-agents,triplicate cultures were washed twice with Tris-buffered saline(20mM Tris-HCl,pH8.0,137mM NaCl)and then treated with lysis buffer containing1%Nonidet P-40,20mM Tris-HCl,137 mM NaCl,0.5mM EDTA,10%glycerol,1mM phenylmethylsulfonyl fluoride,0.15unit/ml aprotinin,20mM leupeptin,and1mM sodium vanadate,at4°C for20min).Insoluble material was removed by centrifugation for10min at12,000g.Triplicate lysate aliquots were diluted in SDS sample buffer(0.06mM Tris-HCl,pH6.8,12.5% glycerol, 1.25%SDS,5%-mercaptoethanol,0.002%bromphenol blue),boiled for5min in SDS sample buffer,and subsequently electrophoresed through7.5%SDS-PAGE.The proteins on the gel were electrotransferred to nitrocellulose.Blots were probed over-night at4°C with the different anti-ERK antibodies and analyzed, using the enhanced chemoluminescence system and horseradish per-oxidase-coupled secondary antibody.After visualization on film,they were quantified by densitometry.Visualization of ERK by Confocal Laser Scanning Fluorescence MicroscopyPC12cells were plated onto collagen-and poly(lysine)-coated two-well chambers(Nalgene)1day before each experiment.The cells were washed with PBS and then incubated for2h at4°C with rabbit anti-phospho ERK antibody diluted1:10,000in PBS containing10% fetal bovine serum and0.2%Triton X-100.The cultures were then washed three times,10min each,at room temperature with0.2% Triton X-100in PBS containing10%fetal bovine serum.The cultures were then incubated with a goat-anti-rabbit IgG-conjugated with Cy3for1h at37°C.The cells were washed twice with1ml of fresh medium and used immediately.Cells were treated with PX(5M) for1to30min or left untreated.The fluorescence of Cy3was quantified with the aid of confocal laser scanning fluorescence mi-croscopy(Leica TCS-4D;Leica Lasertechnik,Heidelberg,Germany), using excitation and emission wavelengths of535and570nm,re-spectively.Gray scale images with0to255steps were collected at different time points before and up to30min after the addition of PX by using a512ϫ512pixel format and archived as image files for quantitative analysis.The intensity of the fluorescence in the indi-vidual cells was measured using Leica quantitation software.Back-ground levels of immunoreactivity were very low in the control cells and this level of nonspecific signal was subtracted from the digitized images,measured in the PX-treated cells.StatisticsImages obtained on X-ray film following autoradiography or chemiluminescence were scanned and the protein bands were quan-tified by densitometry,using NIH Image software.Significant sta-tistical differences between band intensities were determined by ANOVA analysis(pՅ0.05).Also,the significance of the fluorescence value,obtained by confocal microscopy was analyzed by ANOVA(pՅ0.01).The significance of the statistical differences between the results obtained from DA release and vesicle fusion-based ELISA were analyzed by Kruskal-Wallis and Dunn tests(pՅ0.05).ResultsPardaxin-Induced Activation of ERKs in PC12Cells. Using anti-phospho ERKs antibody we observed strong stim-ulation of ERK1(p44MAPK)and ERK2(p42MAPK)by NGF (Fig.1A,top)and to a lesser extent by osmotic shock with sorbitol(Fig.1A,top).The same NGF-and sorbitol-treated cell lysates were also analyzed using immunoblotting with an anti-pan ERK antibody to confirm that the amount of ERK706Bloch-Shilderman et al.enzyme was the same in each sample (Fig.1A,bottom).It is evident that in the absence of NGF and sorbitol both ERK1and ERK2were not active.It can also be seen that other ERKs besides ERK1and ERK2did not cross-react with the anti-phospho ERK antibody.NGF-and sorbitol-treated sam-ples were immunoprecipitated and the activation of the ERKs was evaluated by measuring ERK activity,using MBP as substrate.Results from these experiments were consistent with those obtained by Western blot,and indicated a similar fold increases in phosphorylation (an increase of 13-fold by NGF,and of 6-fold by sorbitol;data not shown).To examine the effect of PX on ERK activity,the cells were treated with NGF (a known stimulator of ERKs),or PX,or were left untreated (CON).The basal activity of ERK1and ERK2was very low in untreated cells (Fig.1B,CON).Treatment of the cells with NGF for 5min caused about 16-fold stimulation of ERK1and ERK2activity (Fig.1B,NGF).As shown in Fig.1B,PX stimulated ERK activity in a time-dependent man-ner.Treatment of the cells with PX for 15min resulted in a maximal ERK stimulation (5-fold increase over basal,Fig.1B,PX).Thereafter (30–60min),PX-stimulated ERK activ-ity gradually decreased,disappearing within 120min to less than control (Fig.1B).The total amount of protein detected using anti-pan ERK antibody did not change throughout the incubation period (data not shown).To evaluate the role of extracellular Ca 2ϩin PX-stimula-tion of ERK activity,PC12cells were treated with PX (4M),for 15min,in the absence of Ca 2ϩ(ϩ1mM EGTA)or in Ca 2ϩ-containing media.The cells were then lysed and West-ern blotted with anti-phospho ERK antibody (Fig.2I),anti-pan ERK (Fig.2II),or phosphorylation experiments were performed with MBP (Fig.2III).In the presence of extracel-lular Ca 2ϩ,the basal activity of ERK1and ERK2was low (Fig.2I,CON).Treatment of the cells with NGF or PX stim-ulated ERK activity by 13-and 5-fold,respectively (Fig.2I,NGF,PX).Measurement of MBP phosphorylation following cell treatment with NGF or PX for 15min showed 5-to 7-fold increased ERK activity,compared with that of the control (Fig.2III,ϩCa 2ϩ).These data further support the activation of ERKs by NGF and PX in Ca 2ϩ-containing medium.In the absence of extracellular Ca 2ϩbasal ERK activity was unde-tectable.Under these conditions,NGF and PX stimulation of ERK activity was very low (Fig.2I)and visualized only upon 24-h exposure of the autoradiograms (data not shown).When the cultures were treated with NGF or PX in Ca 2ϩ-free me-dium,the total amount of ERK1and ERK2proteins,detected with anti-pan ERK antibody (Fig.2II),did not change.A similar lack of ERK stimulation by PX in the absence of extracellular Ca 2ϩwas verified by measuring MBP phos-phorylation (Fig.2III).These data indicate that the loss of ERK activity in the absence of extracellular Ca 2ϩis due to the inability to stimulate ERK by NGF and PX,but not to a reduction in ERK protein level (Fig.2II).Another accepted criterion of ERK activation by growth factors is their ability to translocate to the nucleus.To verify the ability of PX to cause translocation,PC12cells were treated with PX and reacted with anti-phospho ERK followed by fluorescent second antibody,and analyzed after 1-and 30-min treatment by confocal laser scanning fluorescence microscopy (Fig.3).PC12cells at phase contrast showed a distinct nucleus containing nucleoli surrounded by cytoplasm (Fig.3I).After 1-min incubation with PX (Fig.3II),a large amount of active ERK was observed in the cytoplasm.After 30min,the majority of the active ERK was localized in the nucleus (Fig.3III).The most plausible interpretation of these data is that treatment of the cells with PX for 30min resulted in the translocation of the phosphorylated ERKs to the nu-cleus (Traverse et al.,1992).Relationship between the Inhibitory Effect of PD98059and UO126on ERK Activity and PX-Induced Dopamine Release.Recently,it was found that PX induces in a time-dependent manner DA release from PC12cells,Fig.1.Pardaxin stimulation of ERK activity in PC12cells.A,PC12cells were treated (ϩ)for 5min with 50ng/ml NGF or 0.5M sorbitol,or left untreated (Ϫ).The cells were then collected and 10-g protein aliquots were analyzed by SDS-PAGE (10%gel,reducing conditions)and electro-transferred to nitrocellulose membranes.The membranes were probed with anti-phospho (anti-active)ERK antibody or,after stripping,re-probed with antibody for unphosphorylated ERK.NGF (lane 2)and sorbitol (lane 4).Controls (lanes 1and 3).Top,anti-phospho ERK anti-body;bottom,anti-ERK antibody.The location of ERK1and ERK2is indicated by arrows.B,time course of ERK stimulation by 5M PX.Cells were collected and treated as described above.CON,control.NGF,PC12cells were treated for 5min with 50ng/mlNGF.Fig.2.Effect of extracellular calcium on ERK stimulation.PC12cells were treated for 5min with 50ng/ml NGF or for 15min with 5M PX or left untreated (CON).The cells were then collected and 10-g protein aliquots were analyzed by SDS-PAGE (10%gel,reducing conditions)and electrotransferred to nitrocellulose membranes.The membranes were probed with anti-phospho ERK antibody (I)or,after stripping,reprobed with antibody for unphosphorylated ERK (II).ϩCa 2ϩ,calcium (1.8mM)-containing medium.ϪCa 2ϩ,calcium-free medium.I,anti-phospho ERK antibody;II,anti-ERK antibody;III,phosphorylated MBP was visualized by autoradiography,as described under Experimental Procedures .The location of ERK1and ERK2is indicated by arrows.Involvement of ERK in Pardaxin-Induced Dopamine Release707both in the presence and absence of extracellular Ca 2ϩ(Abu-Raya et al.,1999),as well as activation of ERK (Fig.1).Therefore,we examined the relationship between DA release and ERK activation.PC12cells were pretreated with the selective MEK inhibitor PD98059(50M)for 45min.The cells were then stimulated with NGF (50ng/ml)or PX (4M)or left untreated (Fig.4).PD98095completely blocked the basal activity of ERK1and ERK2.Both NGF-and PX-in-duced ERK activity was inhibited by 60and 100%,respec-tively (Fig.4).In the presence of extracellular Ca 2ϩthecomplete inhibition of PX-induced ERK activity was accom-panied by 83and 53%inhibition of PX-induced DA release by PD98095and UO126,respectively (Fig.5A).However,both MEK inhibitors did not affect significantly PX-induced DA release in Ca 2ϩ-free medium (Fig.6).To further investigate the involvement of ERKs in neurotransmitter release,we designed an exocytotic assay,which quantifies vesicular re-lease,reflected by exposure of the vesicular protein synapto-tagmin to the extracellular matrix,using vesicle fusion-based ELISA (Parnas and Linial,1998).As shown in Fig.5B,PX increased synaptotagmin exposure on the cell surface by50%Fig.5.Effect of PD98059and UO126on pardaxin-induced neurotrans-mitter release in Ca 2ϩ-containing medium.A,PC12cells (1ϫ106cells/well)preloaded with [3H]dopamine were treated for 45min with 50M PD98059(PD),or for 30min with 30M UO126,or left untreated.The cells were then exposed to 4M PX for an additional 20min or left untreated (control).DA release was measured as described under Exper-imental Procedures .The values presented are the mean ϮS.E.M.of three independent experiments (n ϭ3in each experiment).*p Ͻ0.05compared with the control (basal release).**p Ͻ0.05compared with PX alone.B,exocytosis was measured by vesicle fusion-based ELISA,as described under Experimental Procedures .Control (untreated)cells or cells pre-treated for 45min with 50M PD98059(PD)were exposed to 4M PX or left untreated for an additional 20min in the presence of anti-synap-totagmin I antibody.The cells were washed,fixed,and incubated with blocking solution.Blue color formation was monitored at OD 450nm,with the aid of an ELISA reader.The values presented are the mean ϮS.E.M.of three independent experiments (n ϭ6in each experiment).*p Ͻ0.05compared with the control (basal release).**p Ͻ0.05compared with PXalone.Fig.6.Effect of PD98059and UO126on pardaxin-induced neurotrans-mitter release in Ca 2ϩ-free medium.PC12cells (1ϫ106cells/well)pre-loaded with [3H]dopamine were treated for 45min with 50M PD98059(PD),or for 30min with 30M UO126,or left untreated.The cells were then exposed to 4M PX for an additional 20min or left untreated (control).DA release was measured as described under Experimental Procedures .The values presented are the mean ϮS.E.M.of three inde-pendent experiments (n ϭ3in each experiment).*p Ͻ0.05compared with the control (basalrelease).Fig.3.PX-induced translocation of active ERKs to the nucleus.Phase contrast micrographs of PC12cells before treatment (I)and immunoflu-orescence micrographs of the same cells treated with PX for 1min (II)or 30min (III).The cells were stained with anti-phospho ERK antibody and detected by the fluorescent CY3dye.Fig.4.Effect of the MEK inhibitor PD98059on NGF and PX stimulation of ERK.To measure ERK activity,cells were pretreated for 45min with 50M PD98059(PD)or left untreated.The cultures were then exposed to 50ng/ml NGF or 5M PX for an additional 20min,or left untreated (CON).Cell lysates (25g/sample)were separated,blotted,and analyzed,using anti-phospho ERKs antibodies,and further processed,as described in the legend to Fig.1.The location of ERK1and ERK2is indicated by arrows.708Bloch-Shilderman et al.。
有机人名反应VilsmeierHaack甲酰化反应
Vilsmeier-Haack reaction
Now
• The application of Vilsmeier-Haack reaction in the synthesis of the agrochemicals such as fenpyroximate,imazaquin,furametpyr was reviewed
Vilsmeier-Haack reaction
Vilsmeier-Haack reaction
References
1 Vilsmeier,A.and Haack,A.Ber.1937,60,119. 2 Campaigne,E.and Archer W. L. Organic Syntheses, Coll.1963,Vol.4,p.331.1953,Vol.33,p.27. 3 Youssefyeh,R.D.Tetrahedron Lett.1964,5,2161. 4 Minkin,V.I.;Dorofeenko,G.N.Russian Chem. Commun.1960,599.(综述) 5 Arnold,Z.;Zemlicka,mun.1959,24,2385. 6 Stéphanie Hesse and Gilbert Kirsch Tetrahedron Lett.2002,43,1213.
Vilsmeier-Haack reaction
• 维尔斯迈尔-哈克反应(Vilsmeier-Haack reaction)是指芳香化合物与二取代甲酰胺在三 氯氧磷作用下,反应生成芳环上甲酰化产物[1]。 该反应只reaction
Vilsmeier-Haack reaction
Vilsmeier-Haack reaction
依达拉奉注射液联合Rho激酶抑制剂对急性脑梗死患者神经功能及血清ENA78、HMGB1水平的影响
中国实用医刊2019年1丿]第46卷第2期Chinese Journal of P ractical Medicine.Jan.2019,Vi)l.4&,2・111・(1):102-105.曹明英,姚朱华,蔡洪滨,等.强化他汀治疗对老年ACS患者PC1围手术期发牛.造影剂肾病危险因素的「•预「•广东保学,2014,35(15):2431-2434.D()l:10.3969/j.issn.1001-9448.2014.15.049.[9]闫婷婷,刘维.杨丽.等•他汀类药物的肾脏安全性及在慢性肾脏病患者中的应用J・药物不良反应杂志,2016.18(5):356-359.1)01:1().3760/cma.j.issn.1008-5734.2016.05.007.JO:李东韬.郑建勇,赵力,等•血NGAL和IL-18对造影剂肾病早期肾损伤的诊断价ffi J.西南国防医药,2015,25(4):349-351.1)01:10.3969/j.issn.1004-0188.2015.04.001.11:赵凯.李永健.泮罗布考对老年不稳定型心绞痛患昏对比剂肾损害的预防作用[J]•重庆医学,2013,42(14):1593-1594.D01:10.3969/j.issn.1671-8348.2013.14.011.12]乔英.杨世诚,付乃宽.*乙酰半胱氨酸对老年冠心病患者介入治疗后造影剂肾病的预防作用」「•中华老年心脑血管病杂志,2013,15(4):339-342.1)01:10.3969/j.issn.1009-0126.2013.(M.(X)2.(收稿H期:2018-11-21)(本文编辑:杨帆)依达拉奉注射液联合Rho激酶抑制剂对急性脑梗死患者神经功能及血清ENA-78.HMGB1水平的影响郭云飞山西省大同市第二人民医院03700()通信作者:郭云飞,Email:j6w44q@163.corn【摘要】目的探讨依达拉奉注射液联合Rh()激酶抑制剂法舒地尔对急性脑梗死患者神经功能及血清中性粒细胞激活肽-78(ENA-78)、高迁移率族蛋白l(HMGBl)水平的影响方法选取大同市第二人民医院2015年12月至2018年3月急性脑梗死患者86例,随机分为两组,每组43例对照组采用依达拉奉治疗,研究组在对照组基础上加用Rho激酶抑制剂(法舒地尔)治疗,均治疗2周比较两组治疗前及疗程结束后日常生活能力(BI)及神经功能抉损(NIHSS)评分、临床疗效、治疗前及疗程结束后血清ENA-78及HMGB1水平、神经生长因子(NGF)、脑源性神经营养因子(BDNF)水平、不良反应发生率结果疗程结束后两组NIHSS.BI分值较治疗前改善,且研究组BI分值高于对照组,NIHSS分值低于对照组(P<0.05);研究组总有效率(90.70%)高于对照组(74.42%,P<0.05);疗程结束后研究组血清ENA-78及HMGB1水平低于对照组,BI)NF、NGF水平高于对照组(P<0.05);研究组不良反应发生率(13.95%)与对照组(9.30%)比较差异未见统计学意义(/〉>0.05)_结论依达拉奉注射液联合Rho激酶抑制剂法舒地尔治疗急性脑梗死患者,可有效降低血清ENA-78及HMGB1水平,提高BDNF及N(;F含量,促进患者神经功能及日常生活能力恢复,提高疾病治疗效果,且不会增加不良反应发生风险,具有一定安全性【关键词】依达拉奉注射液;Rho激酶抑制剂;急性脑梗死;神经功能;中性粒细胞激活肽;高迁移率族蛋白1 DOI:10.3760/cma.j.issn.1674-4756.2019.02.034Effects of edaravone injection combined with Rho kinase inhibitor on neurological function and serum ENA-78,HMGB1levels in patients with acute cerebral infarctionGuo YurifeiThe Second People's Hospital of Datong,Datong037000,ChinaCorresponding author:Guo Yunfei,Email:j6u44(i@[Abstract]Objective To investigate the effects of edaravone injection combined with Rho kinase inhibitor fasudil on neurological function and serum levels of neutrophil activating peptide78 (ENA-78)and high mobility group protein1(I1MGB1)in patients with acute cerebral infarction. Methods Eighty-six patients with acute1cerebral infarction in the Second People's Hospital of Datong from December2015to March2018were enrolled.The patients were randomly divided into two groups, with43cases in each group.Edaravone was used in lhe control group,and the study group was treated with Rho kinase inhibitor(fasudil)on the basis of the treatment in the control group,and both the two・112・中国实用医刊2019年1月第46卷第2期Chinese Journal of Practical Medicineman.2019,Vol.46,No.2groups were treated for2weeks・The daily living ability(BI)and neurological deficit(NIHSS)scores,clinical efficacy,serum ENA-78and HMGB1levels,nerve growth factor(NGF),brain-derived neurotrophic factor(BDNF)and the incidences of adverse reactions were compared.Results Aftertreatment,the NIHSS and BI scores of the two groups were improved in comparison with those beforetreatment,and the BI score of the study group was higher than that of the control group,and the NIHSSscore was lower than that of the control group(P<0.05).The total effective rate of the study group(90.70%)was higher than that of the control group(74.42%,P<0.05).After treatment,the serumlevels of ENA-78and HMGB1in the study group were lower than those in the control group,and thelevels of BDNF and NGF were higher than those in the control group(P<0.05);there was nosignificant difference in the incidence of adverse reactions between the study group(13.95%)and thecontrol group(9.30%,P>0.05)・Conclusions Edaravone injection combined with Rho kinaseinhibitor fasudil for acute cerebral infarction can effectively reduce the serum ENA-78and HMGB1levels,increase the BDNF and NGF levels,and promote the neurological function and daily livingability・It can improve the therapeutic effect of the disease,and does not increase the risk of adversereactions,and has a certain degree of safety・[Keywords]Edaravone injection;Rho kinase inhibitor;Acute cerebral infarction;Neurologicalfunction;Neutrophil activating peptide78;High mobility group protein1DOI:10.3760/cma.j.issn.1674-4756.2019.02.034急性脑梗死为临床多发脑血管疾病,其发病率占脑血管疾病总发病率的75%左右,对患者身心健康及生活质量造成了巨大威胁一‘】。
[精品]生殖内分泌学
下視丘
GnRH(性腺刺激素釋放激 素)→FSH,LH
TRH(甲狀腺刺激素釋放激素) →TSH
CRH(腎上腺皮質素刺激素釋 放激素) →ACTH
GHRH(生長激素刺激素釋放 激素) →GH
PRH(泌乳刺激素釋放激素) PIH泌乳刺激素抑制激素) MRF(黑色素刺激素釋放因子) MIF(黑色素刺激素抑制因子)
催產激素 (Oxytocin)
刺激子宮平滑肌的收縮 促進乳腺管平滑肌上皮的收
縮將乳汁排出
催產激素 (Oxytocin)的作用
GnRH
GnRH的神經元位 於中底下視丘 (MBH)的弓狀神經 核與前下視丘的 視神經前區
GnRH
GnRH is a small peptide with 10 amino acids with some variation in the amino acid sequence among various mammals.
H+R→GTP
cGMP→activated PKA
Calcium
H+R→calcium channel open→ Ca+++calmodulin(調鈣蛋白)
IP3(肌醇三磷酸)
H+R→G-protein→phospholipase C(PLC) →
PIP2 IP3+DAG
DAG(二乙硫甘油脂)
Endocrine 的作用機轉
Endocrine 的作用機轉
Endocrine 的作用機轉
Amines類激素
水溶性 Receptor 在細胞膜內 Primary messenger→secondary messenger
cAMP(環單磷酸腺甘酸) cGMP(環單磷酸鳥甘酸) Calcium IP3(肌醇三磷酸) DAG(二乙硫甘油脂)
葡聚糖激活免疫反应流程
葡聚糖激活免疫反应流程英文回答:Activation of the immune response by beta-glucans involves a complex series of events that occur at both the cellular and molecular levels. Beta-glucans are polysaccharides that are found in the cell walls of certain fungi, bacteria, and plants. When these beta-glucans are recognized by the immune system, they can initiate a cascade of immune responses.The first step in the activation of the immune response is the recognition of beta-glucans by specialized immune cells called macrophages. Macrophages have receptors on their surface that can bind to beta-glucans. Once the beta-glucans are bound to the receptors, a signal is sent to the inside of the macrophage, triggering a series of intracellular events.One of the key events that occurs is the activation ofa transcription factor called NF-kB. NF-kB is a proteinthat regulates the expression of genes involved in the immune response. Once activated, NF-kB translocates into the nucleus of the macrophage and binds to specific DNA sequences, leading to the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta).These pro-inflammatory cytokines play a crucial role in the immune response. They help recruit other immune cells to the site of infection or injury and stimulate their activation. For example, TNF-alpha can induce the expression of adhesion molecules on the surface of endothelial cells, allowing immune cells to adhere to the blood vessel walls and migrate into the infected or injured tissue.In addition to cytokine production, beta-glucans can also enhance the phagocytic activity of macrophages. Phagocytosis is the process by which macrophages engulf and destroy pathogens. Beta-glucans can bind to specific receptors on the surface of macrophages, activatingintracellular signaling pathways that promote phagocytosis. This enhanced phagocytic activity helps to eliminate the invading pathogens and clear the infection.Furthermore, beta-glucans can also stimulate the production of reactive oxygen species (ROS) by macrophages. ROS are highly reactive molecules that can kill pathogens. By increasing ROS production, beta-glucans can further enhance the antimicrobial activity of macrophages.Overall, the activation of the immune response by beta-glucans involves the recognition of beta-glucans by macrophages, the activation of intracellular signaling pathways, the production of pro-inflammatory cytokines, the enhancement of phagocytosis, and the stimulation of ROS production. These immune responses work together toeliminate pathogens and protect the body from infections.中文回答:葡聚糖激活免疫反应涉及一系列复杂的细胞和分子水平的事件。
荷尔蒙的工作原理英语作文
荷尔蒙的工作原理英语作文The Working Principle of Hormones。
Hormones are chemical messengers that are produced by the endocrine glands in the body. They play a crucial role in regulating various physiological processes, such as growth, metabolism, and reproduction. The working principle of hormones is complex and involves a series of intricate processes that help maintain the body's internal balance.Hormones are produced by the endocrine glands, which include the pituitary gland, thyroid gland, adrenal glands, and pancreas, among others. These glands release hormones into the bloodstream, where they travel to target organs and tissues to exert their effects. Hormones can have a wide range of effects on the body, including stimulating or inhibiting the production of other hormones, regulating metabolism, and controlling growth and development.One of the key features of hormones is theirspecificity. Each hormone is designed to interact with specific receptors on target cells, which allows them to exert their effects in a precise and controlled manner. For example, insulin is a hormone that helps regulate blood sugar levels by binding to insulin receptors on cells in the liver, muscle, and fat tissues. This triggers a series of signaling pathways that promote the uptake of glucose from the bloodstream into these cells, lowering blood sugar levels.Hormones can also act in a feedback loop to regulate their own production. For example, the hypothalamus in the brain releases a hormone called corticotropin-releasing hormone (CRH) in response to stress. CRH then stimulates the pituitary gland to release adrenocorticotropic hormone (ACTH), which in turn signals the adrenal glands to produce cortisol. Once cortisol levels in the blood reach a certain threshold, they signal the hypothalamus and pituitary gland to stop producing CRH and ACTH, respectively, thus shutting down the stress response.In addition to their role in regulating physiologicalprocesses, hormones can also influence behavior and mood. For example, the hormone oxytocin is known as the "love hormone" because it is released in response to social bonding and physical touch. Oxytocin promotes feelings of trust, empathy, and connection with others, and is thought to play a role in strengthening social relationships.Overall, hormones play a vital role in maintaining the body's internal balance and ensuring that all physiological processes function smoothly. By acting as chemical messengers, hormones help coordinate the activities of different organs and tissues, allowing the body to respond to changes in the environment and maintain homeostasis. Their intricate working principle highlights the complexity of the human body and the importance of hormonal regulation in overall health and well-being.。
海带多糖拮抗肾上腺素致血管内膜接触性损伤的作用研究
海带多糖拮抗肾上腺素致血管内膜接触性损伤的作用研究陈蒙华;杨晓梅;谢露;肖素军【摘要】Objective:To study the antagonistic effects of polysaccharide from Laminaria on the epinephrine contact injury and the tissue factor (TF) expression on the endangium of rabbits. Methods: To establish a endangium injury model induced by epinephrine on the femoral artery of rabbits. The rabbits were divided into five groups:saline, epinephrine-saline mixture, high, low dose of Laminaria-epinephrine mixture, and enoxaparin-epinephrine mixture was injected respectively to the dissociated femoral artery in the corresponding rabbit groups, and the solution was keeped in the arteries for 25 min. The drug contact arterial segments were conventional sectioned and stained with HE for the observation of the integrality of arterial endangium. The TF immunohistochemistry was done for the demonstration of TF expression in endothelium and subendothelial tissue with mean gray value which is reverse proportional to TF dying. Results: HE stained slices of femoral artery showed that endangium was intact and smooth in saline group, faulted with missing endothelium section in epinephirne group, intact at large with local missing endothelium in low dose Laminaria group, intact with local separation of endothelium and subendothelial tissue in high dose Laminaria group, intact with local roughness in enoxaparin group. The TF immunohistochemistry showed that mean gray value in low dose Laminaria group(68.75±13.21), high dose Laminaria group(85.00±7.37) andepinephrine guoup (105.44 ± 15. 15)were greater than that in epinephrine group(41.12 ± 10.12,P <0. 01). Conclusion:Polysaccharide from Laminaria can protect the integrality of vessel endthangium and reduce the TF expression.%目的:研究海带多糖拮抗肾上腺素致血管内膜接触性损伤、减少组织因子(TF)表达的作用.方法:建立肾上腺素致家兔股动脉内膜接触性损伤模型,将家兔分成5组,分别通过插管把生理盐水、肾上腺素-生理盐水液,低剂量、高剂量海带多糖-肾上腺素液和依诺肝素-肾上腺素液注入相应家兔组的游离股动脉中,保留25 min.取与药物接触的股动脉段,常规切片,HE染色,观察血管内膜的完整情况;将切片作TF免疫组化,以血管平均灰度值反映TF在内皮层和内皮下组织的表达情况,灰度值与TF染色成反比.结果:股动脉血管内膜HE染色切片显示,生理盐水组内膜完整、光滑;肾上腺素组内皮层脱落、缺失;海带多糖低剂量组大体完整,内皮局部脱落;海带多糖高剂量组内皮层完整,局部可见与皮下组织分离;依诺肝素组较完整,局部表面粗糙.TF蛋白免疫组化显示,海带多糖低剂量组(68.75±13.21)、海带多糖高剂量组(85.00±7.37)和依诺肝素组(105.44±15.15)平均灰度值均明显高于肾上腺素组(41.12±10.12)(均P<0.01).结论:海带多糖能够保护血管内膜的完整性,减少TF表达.【期刊名称】《广西医科大学学报》【年(卷),期】2011(028)003【总页数】3页(P329-331)【关键词】海带多糖;肾上腺素;血管内膜;组织因子【作者】陈蒙华;杨晓梅;谢露;肖素军【作者单位】广西医科大学第一附属医院心血管病研究所,南宁530021;广西医科大学生理学教研室;广西医科大学生理学教研室;广西医科大学生理学教研室【正文语种】中文【中图分类】R322.1+2组织因子(Tissue Factor,TF)是一种单链跨膜糖蛋白,是活化凝血因子VII(VIIa)与凝血因子VII(FVII)的受体。
乙酰胆碱受体
②提取膜蛋白
经过许多溶解膜蛋白的尝试,最后发现TritonX100的效果最佳。
TritonX-100是一种非离子型去污剂。它与其他去 污剂的相同点是都具有一个长的能取代膜蛋白上 磷脂的疏水基团,以及一个能够溶解于水的亲水 末端;不同的是大多数其它去污剂都带有一个带 负电的羟基基团,而它没有,所以是非离子型的, 对蛋白质结构的破坏性小。
乙酰胆碱受体
当时Nachmansohn正在研究乙酰胆碱酯酶 (AChase),AChase可酶解从运动神经 末稍释放的ACh。
Nachmansohn知道这类鱼的EO与骨骼肌是 同源的,于是在博览会结束后,开始对EO 进行研究。
对EO的第一次实验结果表明它是AChase 的超级储存库。此器官也是nAChR十分丰 富的储存库,nAChR存在于骨骼肌细胞的 突触后膜上,它会与由运动神经末稍释放 的ACh分子结合。
乙酰胆碱受体
ethane n.乙烷 spray vt.喷射 grid n. 格子, 栅格 peripheral adj.外围的 agrin n.集聚蛋白,集聚素[由运动神经分泌
并可诱 导肌纤维的乙酰胆碱酯酶和乙酰 胆碱受体发生聚集] axon n.轴突 a cascade of events n.级联放大反应
乙酰胆碱受体
这个实验表明,神经并不直接作用
于肌肉,而是通过释放化学物质来
起作用,一号心脏的迷走神经受刺 激时产生了某些物质,它们溶解在
盐水里,对二号心脏产生了作用。
Loewi把这种能抑制蛙的心脏跳动的物质称 作“Vagusstoff” 。 经过几年的研究,Loewi发现这种物质的化 学及生理学性质与乙酰胆碱完全相同,于 是他断定是迷走神经末稍释放的是乙酰胆 碱(ACh) 。
阿魏酸钠经RhoA和Rho-kinase信号通路抑制小鼠肝纤维化的机制研究
阿魏酸钠经RhoA和Rho-kinase信号通路抑制小鼠肝纤维化的机制研究赵蔚林;李君【期刊名称】《医学理论与实践》【年(卷),期】2023(36)3【摘要】目的:探讨阿魏酸钠对于四氯化碳诱导的小鼠肝纤维化的抑制机制。
方法:将CL级昆明属小鼠随机分为正常对照组、CCl4-花生油模型组(模型组)、阿魏酸钠给药组(给药组),每组20只。
模型组小鼠和给药组小鼠分别给予CCl4-花生油(1∶1,V/V)1ml/kg灌胃,正常对照组小鼠给予同等剂量生理盐水,三组均为1次/d,连续8周;从第9周开始,给药组小鼠给予阿魏酸钠20mg/kg灌胃,1次/d,连续给药4周。
12周后,检测各组小鼠肝功能水平、肝影像学指标、病理变化及肝脏中RhoA、Rho-kinase的总体情况。
结果:通过正常对照组、模型组及给药组的小鼠肝功能和肝纤维化指标等指标对比,发现阿魏酸钠能显著抑制小鼠肝纤维化程度。
结论:阿魏酸钠可能通过调节RhoA和Rho-kinase信号通路抑制小鼠肝纤维化。
【总页数】5页(P361-364)【作者】赵蔚林;李君【作者单位】湘潭医卫职业技术学院【正文语种】中文【中图分类】R33【相关文献】1.复方牛胎肝提取物抑制小鼠转化生长因子β1/Smad2信号通路抗肝纤维化的作用机制2.抑制HIF-1α表达对糖尿病小鼠RhoA/ROCK信号转导通路的影响3.阿魏酸钠对肺结核模型大鼠JNK/P38MAPK信号通路的调节作用及巨噬细胞凋亡的抑制作用研究4.抑制G蛋白偶联受体40通过RhoA/ROCK1信号通路缓解小鼠过敏性哮喘5.基于RhoA/ROCK信号通路番茄红素对骨肉瘤MG63细胞荷瘤小鼠的抑瘤作用机制研究因版权原因,仅展示原文概要,查看原文内容请购买。
【PNAS】UCBerkeley科学家发现分子节育新方式
【PNAS】UCBerkeley科学家发现分子节育新方式美国加利福尼亚大学伯克利分校(University of California, Berkeley)科学家发现羽扇豆醇(lupeol)和扁塑藤素(pristimerin),这两种化合物能抑制精子摆动尾部,使其不能向卵子游动并结合。
课题组成员Polina Lishko教授表示,这两种化合物起到了“分子避孕套”的效果,只要很小剂量即可发挥作用,对男女都适用,而且几乎没有副作用。
相关研究成果“Regulation of the sperm calcium channel CatSper by endogenous steroids and plant triterpenoids”在线发表于《美国科学院院刊》(Proceedings of the National Academy of Sciences)。
在最近发表的论文中,UC Berkeley的科学家展示了植物中提取的化学物质如何使精子失去钻入卵细胞的能力:精子有一个特殊的钙离子通道,被称为“Catsper通道”。
当CatSper通道打开,钙离子会进入精子尾部,从而促进尾巴进入一种过度活跃的运动模式。
当精子接近卵子时,Catsper通道接触女性孕酮后,活性大幅上升。
科学家为了阻止精子接近旅程终点时大幅加速的螺旋式运动,对多种化学物质进行了测试筛选。
最终他们发现,羽扇豆醇和扁塑藤素的破坏效果最好。
羽扇豆醇和扁塑藤素并不会损害精子的基本活性,对精子细胞无害,精子还能游动,只是不能有力摆动尾部,因为整个激化通道被关闭了。
目前,该团队正在用灵长类动物的精子试验这种“分子避孕套”的效果。
他们希望能够用这两种化学物质制成紧急避孕药,或制成长期避孕用品。
不过,这种方法现在面临的最大障碍在于制造成本很高,科学家们得先发现可大量提取这两种化学物质的经济途径。
羽扇豆醇存在于存在于羽扇豆种子的表皮中、无花果树和橡胶植物的胶乳中,以及葡萄、橄榄和卷心菜等水果蔬菜中,但含量很低。
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Involvement of Rho Kinase in EndothelialBarrier MaintenanceG.P.van Nieuw Amerongen,C.M.L.Beckers,I.D.Achekar,S.Zeeman,R.J.P.Musters,V.W.M.van HinsberghObjective—Rho kinase mediates vascular leakage caused by many vasoactive agents including thrombin.Enhanced Rho kinase activity induces endothelial barrier dysfunction by a contractile mechanism via inactivation of Myosin Phosphatase(MP).Here,we investigated the contribution of basal Rho kinase activity to the regulation of endothelial barrier integrity.Methods and Results—Using a phospho-specific antibody against the myosin phosphatase targeting subunit (Thr696ϪMYPT1)as a marker for Rho kinase activity,basal endothelial Rho kinase activity was observed at cell-cell contact sites,in vitro and in situ.Thrombin enhanced MYPT phosphorylation at F-actin stress fibers.Inhibition of basal Rho kinase activity for24hours or depletion of Rho kinase(ROCK-I and-II)by siRNA disrupted endothelial barrier integrity,opposite to the previously observed protection from the thrombin-enhanced endothelial permeability.This barrier dysfunction could not be explained by changes in RhoA,Rac1,eNOS,or apoptosis.Remarkably,basal Rho kinase activity was essential for proper expression of the adhesion molecule VE-cadherin.Conclusions—Rho kinase has opposing activities in regulation of endothelial barrier function:(1)an intrinsic barrier-protective activity at the cell margins,and(2)an induced barrier-disruptive activity at contractile F-actin stress fibers.These findings may have implications for long-term antivascular leak therapy.(Arterioscler Thromb Vasc Biol.2007;27:2332-2339.)Key Words:endothelial cellsⅢMYPT1Ⅲmyosin phosphataseⅢcytoskeletonI ncreased endothelial permeability is a vascular reaction to inflammatory and angiogenic stimuli,resulting in vascular leakage.Vascular leakage contributes to the pathogenesis of numerous,often life-threatening,disorders.Excessive plasma extravasation may aggravate acute life-threatening obstruc-tion of respiratory airways during pulmonary disorders.1 Vascular leakage may also cause circulatory collapse in sepsis and contribute to intravitreous scar formation in diabetic retinopathy leading to blindness.2The permeability of tumor vessels is well documented in tumor models and in human cancer,having implications for metastasis.3Remark-ably,few specific therapies are available today to counteract vascular leakage.Cytoskeletal elements play a pivotal role in regulation of endothelial barrier function,principally by determining cell shape,facilitating cell adhesion to subendothelial matrix,and participating in formation of junctional complexes.A major cause of vascular leakage under inflammatory conditions is the loss of endothelial cell(EC)junctional integrity,which is accompanied by the formation of small gaps between ECs. Studies on thrombin-induced endothelial hyperpermeability in vitro have identified at least4independent signaling pathways that contribute to barrier dysfunction:(1)Ca2ϩ-dependent activation of myosin light chain kinase4;(2)a RhoA/Rho kinase-signaling pathway5,6;(3)a protein tyrosine kinase/phosphatase pathway that enhances disruption of in-tercellular junctions7,8;and(4)a new pathway that involves protein kinase C zeta.9During the last decade the central importance of small G proteins in regulating the endothelial barrier function has been established.First,activation of the Rho-like small GTPase RhoA was demonstrated to increase actomyosin contractility,which facilitates the breakdown of intercellular junctions causing barrier dysfunction.5,6A wealth of infor-mation is now available,indicating that its downstream target Rho kinase is involved in endothelial hyperpermeability induced by a variety of vasoactive agents such as VEGF, bacterial toxins,and oxidized LDL.Next,the related Rho-like GTPases Rac1and Cdc42were shown to counteract the effects of RhoA,enforcing the barrier or stimulating barrier recovery respectively.10,11In contrast to its barrier enforcing effects,Rac1was also shown to mediate loss of barrier integrity by vasoactive agents such as VEGF and thrombin, via activation of its downstream target Pak1.12More recently, the barrier-stabilizing properties of cAMP-activated small GTPase Rap1were discovered.13,14These data suggest that aOriginal received January19,2007;final version accepted August7,2007.From the Laboratory for Physiology,Institute for Cardiovascular Research,VU University Medical Center,Amsterdam,The Netherlands. Correspondence to G.P.van Nieuw Amerongen,PhD,VU university Medical Center,Laboratory for Physiology,Boechorstraat7,1081BT, Amsterdam,The Netherlands.E-mail nieuwamerongen@vumc.nl©2007American Heart Association,Inc.Arterioscler Thromb Vasc Biol.is available at DOI:10.1161/ATVBAHA.107.1523222332fine balance in the activities of the distinct small GTPases is essential to proper regulation of endothelial barrier integrity.A striking feature of Rho activation by vasoactive agents is the formation of cytoplasmic F-actin stress fibers(SFs).SFs are long cytoskeletal cables of bundles of F-actin and myosin II/non-muscle myosin filaments,that can contract and exert tension.Myosin-II is believed to be involved in generation of contractile forces.Its activity is mainly controlled by its light chain(MLC-2)phosphorylation,which is regulated by2 classes of enzymes,MLC kinases and myosin phosphatases. MLCK and Rho kinase are the2major MLC kinases,but others exist as well.A type1myosin-associated phosphatase activity has been implicated in the regulation of EC gap formation in vitro,15 and pharmacological inhibitor studies suggested its impor-tance in endothelial contractility.16MP is a holo-enzyme consisting of a catalytic subunit of PP1c-␦of38kDa,a large subunit termed the myosin phosphatase targeting subunit or MYPT1,also known as myosin binding subunit or MBS,of which2isoforms M130/133exist and a20-kDa small subunit of unknown function.17MYPT1is the major regulatory subunit,as it binds both PP1c and phosphorylated myosin-II, thus targeting the substrate MLC-2to the catalytic core of MP.Several kinases are able to phosphorylate-MYPT1and inactivate the MP,including Pak1and Rho kinase.18The expected consequence is enhanced MLC-2phosphorylation and contractility.MYPT1phosphorylation has been demon-strated to occur on stimulation with thrombin in ECs.6,19 Previous studies mainly focused on the role of enhanced Rho kinase activity in hyperpermeability induced by vasoac-tive agents.In the present study,we investigated the contri-bution of basal Rho kinase activity to the regulation of endothelial barrier function.First,the subcellular distribution of Rho kinase activity under basal conditions was compared with the distribution under thrombin-stimulated conditions. Subsequently,the involvement of Rho kinase activity in regulation of basal barrier integrity was investigated.Finally, it was investigated whether inhibition of Rho kinase modu-lated the adherens junctional protein VE-cadherin.Materials and MethodsSources of reagents are listed in the expanded Materials and Methods section in the online data supplement section().Human umbilical vein endothelial cells(HUVECs)were cultured as previously described.7HUVECs with transfected with short interfering(si)RNA duplexes using Amaxa technology. Densitometric analyses of Western blots were performed by AIDA Image Analyzer software.Barrier function was evaluated by the transfer of HRP across HUVEC monolayers grown on polycarbonate filters of the Transwell system.7Alternatively,transendothelial electrical resistance(TEER)was measured.7For3D-Digital fluores-cence imaging microscopy,HUVECs were examined with a ZEISS Axiovert200Marianas inverted microscope.The data acquisition protocol included confocal optical planes to obtain3D definition, followed by a constrained iterative deconvolution operation of the images.Data are reported as meanϮSD.Data were compared by a Student t test.probability values of less than0.05were considered to be significant.ResultsSubcellular Localization of Rho Kinase Activities To measure and visualize Rho kinase activity we used a phosphorylation site-specific antibody against the regulatory subunit of myosin phosphatase(MYPT1).Phosphorylation at T696of MYPT1(phospho-MYPT1)by Rho kinase has been previously reported to inactivate the MP and to serve as a surrogate marker for Rho kinase activity.17Expression of MYPT1in HUVECs was detected by Western blotting,demonstrating a double band ofϷ130kD (supplemental Figure I),in agreement with the previous reported130/133kDa MYPT1isoforms in smooth muscle.17 Quantification demonstrated a3-fold increase in the total amount of phospho-MYPT1after stimulation with thrombin for30minutes(supplemental Figure II).The thrombin-induced phosphorylation of MYPT1was largely prevented by preincubation with the Rho kinase inhibitor Y-27632for30 minutes,indicating that thrombin inhibited global MP activity through Rho kinase.In addition,these data support that phospho-MYPT1serves as a proper surrogate marker for Rho kinase inhibition.To determine the subcellular localization of phospho-MYPT1in HUVECs in detail,we used wide-field3D-deconvolution fluorescence microscopy.High power magni-fication demonstrated a punctate cytoplasmic distribution pattern enriched in perinuclear areas under control conditions, that did not colocalize with the fine cytoplasmic F-actin meshwork ECs(Figure1A and enlargement of its white box in Figure1C;the accompanying line intensity scan is pre-sented at the bottom of panel C).In thrombin-stimulated cells phospho-MYPT1decorated F-actin stress fibers(Figure1B). This suggests that inhibition of MP in thrombin-stimulated cells contributes to contractile properties of stress fibers. As staining for panMYPT1in ECs revealed an intense presence of MYPT1at marginal areas(data not shown),we carefully inspected whether MYPT1was phosphorylated in these areas.In control cells,phospho-MYPT1was visible as a fine peripheral lining(enlargements of the yellow boxes in Figure1E).As can be derived from the line intensity scan (see Figure1E)cortical phospho-MYPT1perfectly colocal-ized with F-actin.To our surprise and in contrast to the global cellular increase in phospho-MYPT1by thrombin(supple-mental Figure II),phospho-MYPT1staining was lower in junctional areas in thrombin-stimulated cells(see enlarge-ment of the yellow box in Figure1F).Quantification con-firmed that thrombin reduced phosphorylation of MYPT1at cell-cell contacts significantly(supplemental Figure III).Of note,thrombin did not influence the total amount of MYPT1 in these areas.To verify these findings in intact vessels,rat renal arte-rioles were isolated,cannulated,and perfused with thrombin. Staining of ECs in situ confirmed the colocalization of phospho-MYPT1with cortical F-actin in control vessels (Figure2,arrow heads).In addition,enhanced MYPT1 phosphorylation associated with central F-actin filaments was observed after exposure of intact vessels to thrombin(Figure 2,arrows).van Nieuw Amerongen et al Dual Role of Rho Kinase in Endothelial Permeability2333To test whether basal Rho kinase activity was indeed respon-sible for phosphorylation of MYPT1in junctional areas,HUVECs were pretreated for 24hours with Y-27632and junctional phospho-MYPT1was quantitated (supplemental Fig-ure IV).Treatment with Y-27632markedly reduced junctional phospho-MYPT1.This was further confirmed by staining for phospho-MLC-2.In accordance with inactivation of MP in those areas,cortical phospho-MLC-2was enriched in resting ECs (supplemental Figure V),and reduced on treatment with Y-27632.Thrombin stimulation enhanced phospho-MLC-2mainly at stress fibers,but reduced cortical phospho–MLC-2.In conclusion,visualization of Rho kinase activity at the subcellular level reveals regional differences in Rho kinase activity.In postconfluent ECs basal Rho kinase activity colocalized with the cortical rim of F-actin,but did not colocalize with the fine cytoplasmic F-actin meshwork.In addition,it reveals opposite regulation by thrombin;thrombin induced a robust Rho kinase activation mainly presentonFigure 1.Subcellular distribution of phospho-MYPT1in control and thrombin-stimulated HUVECs.A and B,Double staining for phospho-MYPT1(green)and F-actin (red)of a control EC shown in panel A and ECs stimulated with 1U/mL thrombin for 30min-utes shown in panel B.Nuclei were stained with DAPI (blue).Yellow boxes represent junctional areas and white boxes cytoplas-mic areas.Boxes are enlarged in panel C-F.Images were obtained by the 3D mode of the microscope;70slices were taken with 0.1m-increments in the z axis.From these stacks,a single optical section with the junctions in focus is shown.Bar,10m.C and D,Phospho-MYPT1in cytoplasmic areas.Images represent enlargements of the indicated white boxes in panels A and B.First row of pictures in panel C and D are raw images straight from the camera,second row represent same images but after application of deconvolution technique to reduce out-of-focus light and improve signal-to-noise ratio.Graphs at the bottom rep-resent line intensities of the indicated lines for F-actin (red)and phospho-MYPT1signals (green).E and F,Phospho-MYPT1in junctional areas.Images represent enlargements of the indicated yellow boxes in panels A and B.For sake of clarity only decon-volved images are presented.Graphs at the bottom represent line intensities of the indicated lines for F-actin (red)and phospho-MYPT1signals (green).2334Arterioscler Thromb Vasc Biol.November 2007F-actin stress fibers,whereas cortical Rho kinase activity was decreased in thrombin-stimulated ECs.Opposite Contribution of Distinct Rho Kinase Activities to Regulation of Endothelial Barrier FunctionAs the data presented in Figure 1point to the presence of intrinsic Rho kinase activity at the periphery of confluent endothelial cells,we wanted to evaluate whether the observed basal Rho kinase activity contributes to endothelial barrier integrity.Therefore,the effect of inhibition of Rho kinase on basal barrier function was studied.HUVECs were seeded on top of porous filters,grown 2days postconfluent,and subsequently preincubated with Y-27632for the indicated time periods.Barrier integrity was evaluated by HRP passage across the monolayers during a 1-hour period.As shown in Figure 3B,30minutes preincubation with Y-27632had no effect on basal HRP passage,whereas 24hours preincubationresulted in a 2-fold increase in HRP passage.Preincubation for 96hours with Y-27632even further increased HRP passage.Alternatively,barrier integrity was evaluated by measure-ment of TEER.24-hour preincubation with Y-27632induced a drop in TEER of 27Ϯ2%(n ϭ4),confirming a decreased barrier function (supplemental Figure VI).24-hour pretreat-ment with the structurally unrelated Rho kinase-inhibitors fasudil (10micromol/L)and H-1152(1micromol/L)induced a similar decrease in TEER (26Ϯ3%,n ϭ4and 17Ϯ5%,n ϭ6).We used siRNAs to target the Rho kinase isoforms ROCK-I and ROCK-II .The efficiency of the transfection was monitored by immunoblotting 48hours after transfection.A net decrease in protein expression of Ͼ75%was observed in HUVECs transfected with the specific siRNA (Figure 4,inset).Targeting both Rho kinase isoforms by siRNA signif-icantly reduced TEER (Figure 4),whereas targetingoneFigure 2.MYPT1phosphorylation in ECs of intact rat arterioles.Immuno-cytochemical staining for phospho-MYPT1(upper row in green)and F-actin (second row in red)or both (third row;yellow indicates colocalization)in ECs in intact arterioles.Nuclei are stained with DAPI (blue).Vessels were stimulated with thrombin (right column)or left untreated (left column).Bar,10m.For identifica-tion purposes,outlines of individual ECs are presented in the lower row.van Nieuw Amerongen et al Dual Role of Rho Kinase in Endothelial Permeability 2335isoform had no effect (ROCK-I )or even elevated TEER (ROCK-II ).In line with previous data,30minutes preincubation with Y-27632reduced the thrombin-induced HRP passage in part(57Ϯ5%,see Figure 3B).The remaining increase in HRP passage reflects the Rho kinase-independent hyperpermeabil-ity response of thrombin (indicated with ⌬in Figure 3B).The Rho kinase–independent increase in endothelial permeability on thrombin stimulation was not affected by preincubation with Y-27632for 24and 96hours.This indicates that Rho kinase–independent aspects of barrier regulation of endothe-lial monolayers were not affected by treatment with Y-27632for prolonged periods.Alterations in RhoA,Rac1,eNOS,and Apoptosis Do Not Explain the Barrier-Disturbing Effects of Rho Kinase InhibitionTo study the mechanism of reduced basal barrier function of Rho kinase inhibitor-treated endothelial monolayers,we first measured activity levels of the Rho proteins RhoA and Rac1.RhoA activity did not change by pretreatment with Y-27632,as was evidenced by G-LISA (0.079Ϯ0.065versus 0.109Ϯ0.061,control versus Y-27632–pretreated cells in arbitrary units,n ϭ3,P ϭ0.591).Rac activity was measured by pulldown-assay,and did not change either (0.98Ϯ0.10versus 1.26Ϯ0.25,control versus Y-2763-pretreated cells,n ϭ6,P ϭ0.332).Second,we wondered whether altered eNOS expression could explain the observed barrier dysfunction.Inhibition of Rho kinase previously was reported to interfere with eNOS protein expression,20and altered eNOS activity results in alterations of barrier function.10However,eNOS expression as evidenced by Western blotting did not change significantly by inhibition of Rho kinase with Y-27632(94Ϯ25%of control;mean ϮSD out of 5independent cultures,P ϭ0.31)and therefore does probably not explain the observed changes in barrier integrity.Finally,we wondered whether enhanced apoptosis could explain endothelial barrier dysfunction.However,no signsofFigure 3.Inhibition of Rho kinase has opposing effects on basal and thrombin-enhanced HRP passage across HUVEC monolayers.A,Schematic representation of experimental protocol:HUVEC mono-layers grown on porous filters were prein-cubated with 10micromol/L Y-27632for 30minutes,24hours,and 96hours as indicated in panel B and subsequently a HRP transfer assay was performed in con-trol and thrombin-stimulated monolayers in the absence or presence of Y-27632.HRP passage during a 1-hour period was measured.B,Inhibition of Rho kinaseincreases basal HRP passage and reduces thrombin-enhanced HRP passage.HUVEC monolayers were incubated for the indi-cated time periods with 10micromol/L Y-27632and HRP passage during a1-hour period was subsequently measured under basal conditions (filled bars)and after stimulation with 1U/mL thrombin (hatched bars).Delta represents the thrombin-induced HRP passage thatremains after inhibition of Rho kinase with Y-27632.6determinations in 2different cultures.*P Ͻ0.05basal HRP passage ofmonolayers pretreated with Y-27632for 24or 96hours vs basal HRP passage of control monolayers.#P Ͻ0.05thrombin-enhanced HRP passage of monolayers pretreated with Y-27632for 24or 96hours vs thrombin-enhanced HRP passage of controlmonolayers.Figure 4.Prolonged inhibition of Rho kinase reduces transendo-thelial electrical resistance (TEER).Effect of ROCK siRNAs on TEER.6determinations in 2different cultures.#P Ͻ0.05.Inset:Changes in the expression of ROCK-I and ROCK-II proteins were monitored by immunoblot 48hours after transfection of ECs cells with ROCK-I or ROCK-II siRNA(s).Blot was reprobed with an antibody against ERM proteins to confirm equal loading.2336Arterioscler Thromb Vasc Biol.November 2007enhanced apoptosis were observed by pretreatment with Y-27632as was evidenced by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)as-say (1.9Ϯ1.3%versus 1.3Ϯ0.7%,control versus 24hour.Y-27632-pretreated cells,n ϭ6,P ϭ0.675).Basal Rho Kinase Activity Is Essential for Maintenance of EC JunctionsTo visualize integrity of adherens junctions,endothelial monolayers were stained for the major endothelial adhesion molecule VE-cadherin.VE-cadherin staining showed an in-tact lining at the cell periphery of confluent ECs (supplemen-tal Figure VIIA,left panel).At places where the peripheral membrane of neighboring cells overlapped,VE-cadherin formed a honeycomb-like structure.After inhibition of Rho kinase for 24hours,the peripheral VE-cadherin lining ap-peared thinner,and was no longer continuous at sites where small gaps were formed between ECs (supplemental Figure VIIA,right panel).These findings were not observed after inhibition of Rho kinase for 30minutes (supplemental Figure VIIA,middle panel).Quantitative analysis confirmed that less VE-cadherin was accumulated in junctional areas after Rho kinase inhibition for 24hours (Figure 5),or after targeting both Rho kinase isoforms by siRNA (supplemental Figure VIIIA,right panel).To investigate whether inhibition of Rho kinase affected the total cellular amount of VE-cadherin,VE-cadherin pro-tein expression was measured by Western blotting.Treatment with Y-27632significantly reduced VE-cadherin protein levels by 38Ϯ12%(n ϭ3,P Ͻ0.05;Figure 5).Targeting both Rho kinase isoforms by siRNA similarly reduced VE-cadherin expression,whereas targeting the single isoforms had no effect (supplemental Figure VIIIB).To investigate whether cortical MYPT1forms a complex with the junctional proteins VE-cadherin and -catenin,theseproteins were immunoprecipitated and precipitated com-plexes were analyzed by Western blotting.VE-cadherin and -catenin form a stable complex with each other,but inter-action with MYPT1was undetectable (supplemental Figure VIIB).Also,probing the blot for the ERM proteins ezrin/radixin/moesin,did not reveal a detectable interaction of VE-cadherin with this family of Rho kinase target molecules,known to anchor the cortical F-actin cytoskeleton to the plasma membrane (data not shown).21Taken together,these data indicate that,in addition to its established barrier-disruptive activity,Rho kinase has an unexpected barrier-protective activity under basal conditions,probably via inactivation of MP at the margins of ECs,necessary for proper recruitment of VE-cadherin to junctional areas.DiscussionMajor findings of the present study are that for the first time we demonstrate that Rho kinase has a dual role in regulation of endothelial barrier function with opposite effects:Rho kinase has (1)an intrinsic activity at cell margins that is essential for proper barrier integrity,and (2)an induced activity at stress fibers that mediates cell contraction resulting in barrier disruption.Based on these data and data from literature,we propose that basal Rho kinase activity contrib-utes to barrier integrity by regulating VE-cadherin,whereas enhanced Rho kinase activity induced by vasoactive agents contributes to barrier dysfunction by inducing contractility of cytosolic located F-actin filaments through MP inhibition.To inhibit Rho kinase,we used Y-27632and hydroxyfa-sudil.When tested on a large panel of protein kinases,these inhibitors only inhibited PRK2with similar potency as Rho kinase,22,23excluding other kinases being responsible for the observations of our study.Most importantly,these structur-ally unrelated inhibitors and downregulation of ROCK1/2expression by siRNA approach similarly reduced basal endo-thelial barrier integrity in our experiments.Our data reveal an unexpected cortical activity of Rho kinase in postconfluent ECs.This localized Rho kinase activity reduces MP activity at the margins of the cells,resulting in a peripheral rim of phosphorylated MLC-2.Enhanced peripheral phosphorylation of MLC-2is a pattern also seen when endothelial monolayers are treated with barrier-protective agents such as sphingosine 1-phosphate.24This suggests that those barrier-protective agents enforce a basal active process.These elevated levels of phosphorylated MLC-2spatially localized within cortical F-actin ring might provide an environment with increased tension in junctional areas,which previously has been suggested to contribute to development of junction integrity via enhanced affinity be-tween adherens junctions and the cortical cytoskeleton.24The initial stimulus responsible for basal Rho kinase activity is likely cell–cell interaction,as VE-cadherin engagement recently was shown to activate RhoA in ECs,resulting in tension.25Targeting Rho kinase isoforms by siRNAs revealed that each isoform was dispensible for forming a proper barrier.This suggests that ROCK-I and ROCK-II can functionally replace each other mutually.Targeting both isoformsseverelyFigure 5.Inhibition of Rho kinase interferes with VE-cadherin at cell–cell contacts.Quantitative analysis of VE-cadherin in junc-tional areas.ECs were incubated for the indicated time periods with Y-27632.VE-cadherin accumulation at junctional areas was measured using the line scan function of AIDA software to quantitate fluoresceinisothiocyanate (FITC)fluorescence inten-sity.Values are the mean ϮSD of at least 12cell contacts.*P Ͻ0.05.Inset:representative Western blot showing in theupper panel reduced VE-cadherin protein levels after preincuba-tion with Y-27632for 24hours.Lower panel:the same blot was reprobed with a -actin antibody to verify equal protein loading.van Nieuw Amerongen et al Dual Role of Rho Kinase in Endothelial Permeability 2337disrupted barrier integrity,in line with the effects of the pharmacological inhibitors,all of them inhibiting both iso-forms.Remarkably,single targeting of ROCK-II even im-proved barrier function,suggesting that this is the isoform that is mainly responsible for the barrier disruptive effects. Indeed,it was shown in epithelial cells that ROCK-II,but not ROCK-1mediates disassembly of the junctions.26In ECs ROCK-II,but not ROCK-I has been implicated in micropar-ticle generation.27These specific functions require further investigation.In epithelial cells Rho kinase was shown to be necessary for the local concentration of E-cadherin in cell–cell con-tacts.28In apparent contrast,Braga et al reported that in the endothelial context junctional maturation is not dependent on RhoA activity.29They observed that after inhibition of RhoA for2hours ECs are still able to form new cell–cell contacts. Here,we extend these findings by determining the effects of inhibition of Rho kinase for longer periods on junctional integrity and evaluation of endothelial barrier function.Of note,we chose our conditions such that allowed formation of adherens junctions before we started the Rho kinase inhibitor studies.Detailed analysis reveals a reduced peripherally-localized VE-cadherin expression and an impaired endothe-lial barrier when Rho kinase is inhibited.Several scenarios exist for how Rho kinase activity might contribute to a proper barrier function.Rho kinase results in phosphorylation of ERM proteins via inactivation of MP,and activated ERM proteins anchor the cortical F-actin cytoskel-eton properly to the plasma membrane.21A proper plasma membrane anchorage is essential to develop actomyosin tension,which is required for correct recruitment of adherens junction components.In addition,recent data indicate that ERM proteins can activate Rac1,30and might therefore contribute to Rac1-mediated barrier protection.A more likely scenario,however,is that Rho kinase plays a role in proper recycling of VE-cadherin to EC junctions. The VE-cadherin interaction with the F-actin cytoskeleton has a very dynamic nature.31Reduced VE-cadherin recycling recently was shown to play an important role in VEGF-enhanced endothelial permeability.32Furthermore,Rho ki-nase has been implicated in endosomal trafficking.33There-fore,we propose that impaired VE-cadherin endosomal recycling results in enhanced VE-cadherin degradation in Y-27632–treated cells.The dual regulation of Rho kinase by thrombin has several implications.First,these data provide a warning for the single use of quantitative Western blotting to measure Rho kinase activity by surrogate markers like phospho-MYPT1,as con-current opposite subcellular activities are masked.Second,it indicates that timing and subcellular targeting are important when developing pharmacological agents to inhibit vascular leak.Therefore,our findings warrant attention to the time window for treatment with Rho kinase interfering drugs of patients.Although the negative effects of longer incubations with Rho kinase inhibitors on basal barrier integrity did not outweigh the positive effects in reducing the thrombin re-sponse,these data indicate that—in contrast to what has been thought—inhibition of Rho kinase might negatively influence endothelial barrier function in the long run.In conclusion,those data reveal a dual role for Rho kinase–mediated MP inactivation in the regulation of barrier integrity.AcknowledgmentsWe thank C.Jungerius and M.Van Wijhe for excellent technical assistance.Sources of FundingG.P.v.N.A.was supported by a grant from the Netherlands Heart Foundation(T2003-0032).Our laboratory was supported by the EU (EVGN contract LSHM-2003-503254).DisclosuresNone.References1.Groeneveld AB.Vascular pharmacology of acute lung injury and acuterespiratory distress syndrome.Vascul Pharmacol.2002;39:247–256. 2.Shiels IA,Zhang S,Ambler J,Taylor SM.Vascular leakage stimulatesphenotype alteration in ocular cells,contributing to the pathology of proliferative vitreoretinopathy.Med Hypotheses.1998;50:113–117.3.Weis S,Cui J,Barnes L,Cheresh D.Endothelial barrier disruption byVEGF-mediated Src activity potentiates tumor cell extravasation and metastasis.J Cell Biol.2004;167:223–229.4.Wysolmerski RB,Lagunoff D.Involvement of myosin light-chainkinase in endothelial cell retraction.Proc Natl Acad Sci U S A.1990;87:16–20.5.van Nieuw Amerongen GP,van Delft S,Vermeer MA,Collard JG,vanHinsbergh VW.Activation of RhoA by thrombin in endothelial hyper-permeability:role of Rho kinase and protein tyrosine kinases.Circ Res.2000;87:335–340.6.Essler M,Amano M,Kruse HJ,Kaibuchi K,Weber PC,Aepfelbacher M.Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells.J Biol Chem.1998;273: 21867–21874.7.van Nieuw Amerongen GP,Draijer R,Vermeer MA,van Hinsbergh VW.Transient and prolonged increase in endothelial permeability induced by histamine and thrombin:role of protein kinases,calcium,and RhoA.Circ Res.1998;83:1115–1123.mpugnani MG,Zanetti A,Corada M,Takahashi T,Balconi G,Breviario F,Orsenigo F,Cattelino A,Kemler R,Daniel TO,Dejana E.Contact inhibition of VEGF-induced proliferation requires vascular en-dothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148.J Cell Biol.2003;161:793–804.9.Li X,Hahn CN,Parsons M,Drew J,Vadas MA,Gamble JR.Role ofprotein kinase Czeta in thrombin-induced endothelial permeability changes:inhibition by angiopoietin-1.Blood.2004;104:1716–1724. 10.Mehta D,Malik AB.Signaling mechanisms regulating endothelial per-meability.Physiol Rev.2006;86:279–367.11.Wojciak-Stothard B,Ridley AJ.Rho GTPases and the regulation ofendothelial permeability.Vascul Pharmacol.2002;39:187–199.12.Stockton RA,Schaefer E,Schwartz MA.p21-activated kinase regulatesendothelial permeability through modulation of contractility.J Biol Chem.2004;279:46621–46630.13.Cullere X,Shaw SK,Andersson L,Hirahashi J,Luscinskas FW,Mayadas TN.Regulation of vascular endothelial barrier function by Epac,a cAMP-activated exchange factor for Rap GTPase.Blood.2005;105:1950–1955.14.Fukuhara S,Sakurai A,Sano H,Yamagishi A,Somekawa S,Takakura N,Saito Y,Kangawa K,Mochizuki N.Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1signaling pathway.Mol Cell Biol.2005;25:136–146.15.Verin AD,Patterson CE,Day MA,Garcia JG.Regulation of endothelialcell gap formation and barrier function by myosin-associated phosphatase activities.Am J Physiol.1995;269:L99–108.16.Knapp J,Boknik P,Luss I,Huke S,Linck B,Luss H,Muller FU,MullerT,Nacke P,Noll T,Piper HM,Schmitz W,Vahlensieck U,Neumann J.The protein phosphatase inhibitor cantharidin alters vascular endothelial cell permeability.J Pharmacol Exp Ther.1999;289:1480–1486.2338Arterioscler Thromb Vasc Biol.November2007。