氯化锂降低氧化应激水平改善APP/PS1双转基因小鼠学习记忆功能的机制

目的锂(Li)对神经退行性疾病有潜在神经保护作用,但其分子作用机制尚不清楚。在阿尔茨海默病(AD)发病机制中,氧化应激(OS)水平升高及β-淀粉样肽(Aβ)产生过多是重要的导致患者智力低下的致病因素之一。本课题探讨经氯化锂(Li Cl)处理的APP/PS1双转基因小鼠是否能减少Aβ的生成,以及其是否能通过调控糖原合成激酶-3β(GSK3β)/核因子E2相关因子(Nrf2)/血红素加氧合酶1(HO-1)通路来减轻该小鼠脑组织OS损伤,并逆转APP/PS1双转基因小鼠学习记忆能力水平的降低。方法应用免疫组化方法检测各组小鼠脑组织中老年斑的数量;用PCR法扩增模板DNA进行双转基因突变鉴定,筛选APP/PS1双转基因小鼠;转基因及野生型小鼠随机分为4组,即野生型组、野生型+Li Cl处理组、转基因+Li Cl处理组、转基因组。野生型+Li Cl处理组及转基因+Li Cl处理组小鼠4月或8月龄时每天1次用Li Cl灌胃处理(100 mg·kg~(-1),为期2个月),野生型组及转基因组小鼠则用同等容量的生理盐水进行灌胃处理;蛋白印迹法检测动物脑组织中GSK3β,p-GSK3β(ser9),Nrf2及HO-1蛋白表达水平;用生物化学方法测定各组小鼠脑组织与血清中SOD和GSH-Px活性及MDA含量;ELISA法测定脑组织内不可溶性Aβ含量;尼氏染色检测各组小鼠脑组织神经元尼式小体密度;行为学实验检测各组小鼠学习记忆能力。结果与野生型小鼠相比,6月及10月龄APP/PS1双转基因小鼠脑组织内可见明显的老年斑,不可溶性Aβ含量较高,以10月龄小鼠尤为明显(P<0.05);同时GSK3β活性明显增加,Nrf2和HO-1蛋白表达水平降低,脑组织及血清中SOD和GSH-Px活性降低,MDA含量增加(P<0.05);且转基因小鼠脑组织神经元中尼式小体密度明显减少,其学习记忆能力降低。而对4月或者8月龄APP/PS1双转基因小鼠予以Li Cl进行灌胃处理2月后,与未经处理的同龄APP/PS1双转基因小鼠相比,其脑组织内可见老年斑、不可溶性Aβ含量下降(P<0.05);同时APP/PS1双转基因小鼠脑组织中GSK3β活性、Nrf2及HO-1蛋白表达水平的改变被逆转,转基因动物脑组织和血清OS水平降低(P<0.05),且尼式小体密度明显增加,小鼠空间学习记忆能力提升(P<0.05)。结论 APP/PS1转基因小鼠体内氧化应激水平升高伴随空间记忆力及学习能力下降及脑组织神经元中尼式小体密度减少;Li Cl能减少APP/PS1双转基因小鼠脑组织中Aβ含量,从而减少Aβ在AD发病中的毒性作用,同时Li Cl能预防及逆转APP/PS1双转基因小鼠脑组织中GSK3β/Nrf2/HO-1通路表达的下调,提高机体抗氧化应激能力,减少OS损伤,从而改善APP/PS1双转基因小鼠学习记忆能力,增加尼式小体密度,发挥神经保护作用。     

C反应蛋白对β淀粉样蛋白生成的调节参与Alzheimer’s病发病的研究

β淀粉样蛋白(amyloid-β,Aβ)是阿尔茨海默病(Alzheimer’s disease,AD)发病的重要因素。我们前期发现C反应蛋白(C-reactive protein,CRP)能诱导大鼠产生与Aβ相似的认知障碍,提示CRP可能是AD的重要致病因子。本实验目的是利用PC12细胞初步探讨CRP的细胞毒性与Aβ相关因子淀粉样前体蛋白(amyloid precursor protein,APP),β-分泌酶(β-site of APP cleaving enzyme,BACE-1)、早老素1(presenilins-1,PS-1)和早老素2(presenilins-2,PS-2)的表达调控关系。继而以APP/PS1转基因小鼠作为研究对象,分析脑内CRP和Aβ1-42的相关性;同时脑室给予APP/PS1转基因小鼠CRP以及bis(PC)-H后,观察CRP及其抑制剂是否能影响转基因小鼠的学习记忆,并分析脑内Aβ斑块的形成以明确CRP参与AD早期发病过程的机制。结果显示,CRP诱导PC细胞的相对活力下降,LDH释放增加,表明CRP对PC12细胞产生毒性损伤作用;bis(PC)-H则通过拮抗CRP活性逆转CRP对PC12细胞产生的毒性作用。CRP处理细胞后可诱导Aβ生成相关因子和Aβ1-42的表达显著上调,而bis(PC)-H则可以逆转CRP这一效应,提示了CRP可以调节Aβ的生成。我们在动物实验中发现APP/PS1转基因小鼠脑内CRP与Aβ1-42的水平存在正相关,推测CRP可能通过调节Aβ1-42的生成参与AD早期的发病。我们通过脑室给药的方式给予疾病早期的APP/PS1转基因小鼠CRP及其抑制剂,发现CRP可以影响其学习记忆和Aβ斑块形成。拮抗CRP可以逆转APP/PS1转基因小鼠在物体识别、Morris水迷宫和跳台实验中轻度学习记忆损伤的趋势,并减少Aβ斑块的形成;给予CRP则可以加重APP/PS1转基因小鼠的学习记忆的损伤,并增加Aβ斑块的形成,一正一反地证明了CRP参与AD转基因小鼠的发病。本实验发现了CRP是一个可以作为早期干预AD发病的新靶点,也阐明了CRP参与AD发病的新机制。     

EGCG通过抑制p75~(NTR)通路对APP/PS1小鼠学习记忆障碍的改善作用

目的探讨(-)表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)对APP/PS1转基因小鼠学习记忆障碍的改善作用及其对海马内p75NTR信号通路及其介导的神经细胞凋亡的影响。方法以Morris水迷宫及被动避暗实验观察其行为学变化,TUNEL法及fluoro-Jade B法检测神经细胞凋亡及神经元退行性变,并以Western blot法检测小鼠海马p75NTR相关蛋白的表达水平。结果 EGCG可明显改善APP/PS1转基因小鼠的学习记忆障碍,抑制神经细胞凋亡及退行性改变。同时,EGCG可明显抑制p75NTR通路,降低其剪切产物p75ICD表达及JNK2磷酸化水平,减少p53和cleaved-caspase 3蛋白表达,从而抑制神经细胞凋亡和改善学习记忆障碍,发挥神经保护作用。结论 EGCG通过抑制p75NTR信号通路,抑制神经细胞凋亡,改善学习记忆障碍。     

淫羊藿激活CREB/BDNF信号通路改善APP/PS1双转基因小鼠的认知功能

目的 研究淫羊藿能否通过调节CREB/BDNF信号通路改善APP/PS1双转基因小鼠的认知功能。方法 取7月龄雄性APP/PS1双转基因小鼠30只,按体重随机分为模型对照组和淫羊藿组,每组15只;另取15只同龄的雄性C57BL/6小鼠作为空白对照组。淫羊藿组灌胃给予3 g·kg^-1的药物溶液,空白组和模型组给予等体积0.3% CMC-Na,连续灌胃给药28 d,每天1次。筑巢试验检测小鼠日常生活能力;Morris水迷宫试验检测小鼠的学习和记忆能力;苏木素-伊红（H&E）和尼氏（Nissl）染色法观察小鼠脑组织病理学变化;采用TBA法、羟胺法检测小鼠脑组织中丙二醛（Malondialdehyde,MDA）和超氧化物歧化酶（Superoxide dismutase,SOD）的水平;免疫荧光化学法检测小鼠脑内β-淀粉样蛋白（amyloid β-protein,Aβ）的表达情况;Western blot法检测环磷酸腺苷效应元件结合蛋白（cAMP response element binding protein,CREB）和脑源性神经营养因子( brain-derived neurotrophic factor,BDNF)信号通路相关蛋白的表达情况。结果 与空白组比较,APP/PS1双转基因小鼠的筑巢得分明显降低,逃避潜伏期和游泳距离明显增加（P<0.01）,目标象限停留时间和穿越平台次数显著减少;皮层和海马存在明显的神经病理损伤。此外,APP/PS1双转基因小鼠的皮层和海马区域可观察到大量Aβ沉积,并且脑组织中SOD活性降低,MDA水平升高。Western blot 结果显示,模型组小鼠脑内CREB蛋白磷酸化受到抑制,伴随着BDNF蛋白表达明显降低。淫羊藿（3 g·kg^-1）连续灌胃4周,可明显改善模型小鼠的筑巢能力和认知功能,减轻神经病理损伤,抑制Aβ沉积,减轻氧化应激,促进CREB蛋白磷酸化,增加BDNF蛋白表达。结论 淫羊藿可明显改善APP/PS1双转基因小鼠的认知功能和神经病理损伤,其作用机制可能与激活CREB/BDNF信号通路有关。     

蛇床子素对APP/PS1小鼠学习记忆的影响

目的观察蛇床子素(OST)对APP/PS1小鼠学习记忆功能的影响。方法采用雄性APP/PS1双转基因小鼠及野生型(WT)小鼠,随机分为3组:WT对照组、APP/PS1对照组和APP/PS1+OST。给药组小鼠自9月龄开始每天给予OST10 mg·kg~(-1),对照组小鼠则给予等体积的双蒸水,连续给药12周。每月观察小鼠筑巢行为的改变;实验结束前采用Morris水迷宫观察小鼠行为学的改变。之后,麻醉并处死小鼠,收集脑组织,采用Nissl染色观察海马神经元的形态及数量;Western蛋白印迹法检测海马组织Aβ1-40/Aβ1-42含量,APP、α分泌酶(ADAM10)及β分泌酶(BACE1)的蛋白表达水平。结果 APP/PS1小鼠筑巢行为评分随着时间推移而逐渐变差,不能筑成完整的巢穴;给予OST治疗后,小鼠的筑巢行为评分接近WT小鼠,基本筑成完整巢穴。由此说明,12月龄的APP/PS1小鼠日常生活能力和社会行为有所损伤,而OST对此可部分恢复。水迷宫结果显示,与WT小鼠相比,APP/PS1小鼠出现明显的空间学习记忆能力的下降,表现为平均逃避潜伏期明显延长且目标象限停留时间百分比和穿越平台次数明显减少;给予OST后改善了APP/PS1小鼠的空间学习记忆能力。组织形态及分子生物学结果显示,APP/PS1小鼠海马DG区及CA3区神经元的细胞形态受损并伴有完整神经元数量的减少,并且海马组织Aβ1-40/Aβ1-42含量增加,APP及BACE1蛋白呈现高表达,ADAM10的蛋白表达下降;给予OST后,减少了APP/PS1小鼠海马组织Aβ的含量,降低了APP及BACE1蛋白水平,增加了ADAM10的蛋白表达。结论 OST可改善APP/PS1小鼠日常生活能力、社会行为及空间学习记忆等能力,并减少海马组织Aβ的含量,可能与调节海马APP的酶切途径有关。     

α7 nAChR通过提高抗氧化能力提升AD模型动物学习记忆能力及抗神经毒性作用

目的α7尼古丁型乙酰胆碱能受体(α7 nAChR)对学习与记忆功能密切相关,但其作用机制尚不十分清楚。为此,本研究选用APP/PS1双转基因动物模型,应用α7 nAChR激动剂/抑制剂研究nAChR是否能对抗Aβ引起的神经毒性作用,改善学习与记忆功能。方法选择α7 nAChR激动剂PNU-282987、α7 nAChR抑制剂甲基牛扁碱(MLA)分别处理6月龄、10月龄APP/PS1双转基因小鼠,水迷宫测定小鼠空间学习及记忆能力;HE染色观察APP/PS1双转基因小鼠脑组织病理学改变;免疫组织化学染色检测小鼠大脑内老年斑分布及表达;生物化学方法测定小鼠大脑及血清中丙二醛(MDA)含量、SOD及谷胱甘肽过氧化物酶(GSH-Px)活性;Western蛋白印迹法检测小鼠海马APP代谢途径中α分泌型淀粉样前体蛋白(αAPP)、β分泌型淀粉样前体蛋白(βAPP)、β淀粉样前体蛋白裂解酶1(BACE1)、BACE2及解聚素金属蛋白酶结构域蛋白10(ADAM10),抗氧化应激通路核因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)、突触素(SYN)和突触相关蛋白(SNAP-25)等蛋白的表达水平。结果在6月龄、10月龄小鼠中,与对照组相比,PNU处理组小鼠的平台逗留时间明显升高,穿越平台次数明显增多,逃避潜伏期也有相应缩短;而MLA处理组小鼠平台逗留时间,穿越平台次数明显减少,逃避潜伏期则相应延长。6月龄APP/PS1转基因小鼠中,PNU处理组小鼠脑组织MDA含量较阳性对照组降低,SOD及GSH-Px活性较阳性对照组明显升高;MAL处理组小鼠脑组织MDA含量较阳性对照组升高,而SOD及GSH-Px活性较阳性对照组明显降低。10月龄APP/PS1转基因小鼠中,氧化应激指标略有加重,血清中氧化应激水平变化与脑组织中相似。Western蛋白印迹法结果显示,在6月龄小鼠中,PNU能够上调Nrf2/HO-1,ADAM10,BACE2,αAPP,SYN和SNAP-25蛋白表达水平,MLA可降低BACE1,αAPP,SYN和SNAP-25蛋白表达水平;在10月龄小鼠中,PNU能够上调Nrf2/HO-1,ADAM10,αAPP,SYN和SNAP-25蛋白表达水平,MLA可降低ADAM10,αAPP,SYN,SNAP-25蛋白表达水平。结论α7 nAChR可通过提高抗氧化能力来提升AD动物学习记忆能力及抗神经毒性作用:(1)激活α7 nAChR能改善APP/PS1双转基因小鼠学习记忆能力,而抑制α7 nAChR可加重该动物模型学习记忆障碍;(2)激活α7 nAChR能够活化Nrf/HO-1抗氧化应激通路和降低氧化应激水平;(3)α7 nAChR可调节APP代谢相关酶,促进αAPP生成,抑制Aβ生成及增强SYN、SNAP-25蛋白表达,改善突触可塑性。     

新药AD-08对两种痴呆动物模型学习记忆障碍和海马乙酰胆碱酯酶活性的影响

目的 研究抗阿尔采末病(Alzheimer's disease,AD)新药AD-08对动物学习记忆功能以及海马乙酰胆碱酯酶(AChE)活性的影响.方法 采用侧脑室注射Aβ1-42蛋白的引起的痴呆大鼠模型和APP/PS1双转基因痴呆小鼠模型.SD大鼠分为正常组、模型组、多奈哌齐对照组、AD-08低、中、高剂量组.APP/PS1双转基因小鼠分为模型组、多奈哌齐对照组、AD-08低、中、高剂量组,以及用具有相同遗传背景的小鼠作为正常对照组.正常对照组及模型组动物每天固定时间灌胃给予相应剂量的药物溶剂(生理盐水);阳性药对照组和AD-08各剂量组动物分别给予相应剂量的药物.SD大鼠给药8 d,APP/PS1双转基因小鼠给药1个月,之后用Morris水迷宫方法对动物进行空间记忆能力测试.并用AChE活性试剂盒测定受试动物大脑海马组织的AChE活性.结果 与正常对照组相比,痴呆模型组大鼠和小鼠的空间学习记忆能力均明显降低(P＜0.05),海马组织中的AChE活性明显升高(P＜0.05).各剂量的AD-08均能够明显改善痴呆模型动物的空间学习记忆能力,并降低其海马组织AChE的活性(P＜0.05).所试剂量(0.7 mg·kg-1~10 mg·kg-1)的AD-08改善痴呆模型动物空间学习记忆能力的作用与多奈哌齐(1 mg·kg-1~3 mg·kg-1)相似(P＞0.05).结论 AD-08可以明显改善Aβ1-42所致的痴呆模型大鼠和APP/PS1双转基因痴呆模型小鼠的空间学习记忆能力;其作用机制可能与抑制海马AChE的活性有关.     

新型乙酰胆碱酯酶抑制剂双配基-（-）-美普他酚改善APP/PS1转基因小鼠学习记忆功能的机制

目的阿尔茨海默病(AD)是一种以认知功能障碍为主要特征的神经退行性疾病,是老年痴呆症最常见的类型。目前针对该病已上市的药物多为胆碱酯酶抑制剂,但只能改善AD的认知功能障碍,不能有效干预AD的疾病进程。双配基-(-)-美普他酚[Bis(9)-(-)-meptazinol,B9M]是依据多靶点导向配体策略设计研发的新型胆碱酯酶抑制剂,可同时作用于乙酰胆碱酯酶(ACh E)的催化位点和外周阴离子位点,能够有效抑制ACh E活性及由其诱导产生的β-淀粉样蛋白(Aβ)聚集。本研究主要探讨B9M对AD转基因模型小鼠学习记忆功能的影响,进而分析其潜在的病理机制。方法选用8月龄APP/PS1双转基因小鼠作为AD模型,以3个剂量B9M(每天0.2,0.6和2μg·kg~(-1))皮下注射4周,通过筑巢、新物体识别和Morris水迷宫实验,研究B9M对AD模型小鼠认知功能障碍的改善作用。实验结束后将小鼠处死,收集脑组织样本进行免疫组化和ELISA等实验,研究B9M对AD小鼠脑内ACh E活性、Aβ含量和胶质细胞活化的影响,探讨B9M对AD模型小鼠的疾病修饰作用。结果8月龄APP/PS1转基因小鼠筑巢能力明显下降。B9M经皮下给药4周后,B9M给药组的筑巢评分明显高于未给药APP/PS1模型组,提示B9M能够明显提高小鼠的筑巢能力。APP/PS1转基因小鼠新旧物体识别指数为44.09%,接近50%,提示其对新旧物体无识别能力。而B9M连续给药4周可显著提高APP/PS1小鼠的新物体识别指数,进一步表明B9M能够改善AD模型小鼠的识别记忆能力。水迷宫实验结果表明,B9M给药可明显缩短APP/PS1小鼠寻找平台的潜伏期。与模型组相比,B9M给药组(0.6和2μg·kg~(-1))及阳性药物多奈哌齐组小鼠在目标象限穿越平台时间和距离百分比均存在显著性差异。各组小鼠的游泳速度不存在显著性差异,提示B9M能显著提高小鼠的空间学习和记忆能力。探讨B9M作用机制发现,B9M可通过抑制皮质和海马区ACh E活性降低乙酰胆碱(ACh)水解,提高突触间隙ACh含量。B9M可显著降低APP/PS1转基因小鼠脑内可溶和不可溶性Aβ40和Aβ42含量,显著抑制脑组织中胶质细胞的集聚,减少胶质细胞的增生。B9M 2.0μg·kg~(-1)减少Aβ斑块、抑制胶质细胞活化的效果最为显著,给药剂量是阳性对照药物多奈哌齐的千分之一。结论 B9M皮下连续给药4周可有效提高8月龄APP/PS1转基因小鼠的筑巢和新物体识别能力,改善空间学习和记忆障碍。B9M通过抑制APP/PS1小鼠脑内ACh E活性改善其学习记忆功能,通过减少脑内Aβ斑块、降低Aβ40和Aβ42含量、抑制胶质细胞激活逆转AD相关病理进程,提示B9M具有症状改善和疾病修饰双重作用,是极有潜力的抗AD新药。     

瑞香素对阿尔茨海默病模型小鼠自噬的影响

目的 探讨瑞香素对阿尔茨海默病(Alzheimer’s disease，AD)自噬的影响以及对AD神经保护的作用机制。方法 选用APP/PS1双转基因模型小鼠，3个月龄后开始灌胃给药。药物治疗3个月后，通过Morris水迷宫试验观察瑞香素对AD模型小鼠空间探索和记忆能力的影响；采用ELISA试剂盒检测AD模型小鼠海马脑组织Aβ_1-40、Aβ_1-42水平；利用Western blotting检测瑞香素对AD模型小鼠海马组织微管相关蛋白轻链3Ⅱ(microtubule-associated proteins light chain 3 Ⅱ，LC3-Ⅱ)、beclin-1及p62表达水平的影响。结果 Morris水迷宫试验显示，APP/PS1双转基因模型小鼠逃避潜伏期较对照组明显延长，原象限停留时间明显缩短；瑞香素组小鼠逃避潜伏期较模型组明显缩短，原象限停留时间明显延长。与对照组比较，APP/PS1双转基因模型小鼠脑组织中Aβ水平增多，而瑞香素减少模型小鼠Aβ水平；Western blotting结果显示AD模型小鼠海马区LC3-Ⅱ及beclin-1显著降低，p62显著增加，而瑞香素可上调APP/PS1双转基因模型小鼠LC3-Ⅱ及beclin-1表达并抑制p62的蛋白表达。结论 瑞香素通过上调LC3Ⅱ和beclin-1自噬相关蛋白增强自噬，改善AD模型小鼠的学习记忆功能。     

表没食子儿茶素没食子酸酯对APP/PS1转基因小鼠认知功能和海马自噬的影响

目的探讨表没食子儿茶素没食子酸酯(EGCG)对APP/PSl转基因小鼠海马神经元自噬的作用。方法8月龄APP/PSl转基因小鼠随机分为模型组(Tg)、EGCG低剂量组(Tg/EGCG-L)、高剂量组(Tg/EGCG-H),同月龄C57BL/6J小鼠作为对照组(NT)。采用Morris水迷宫检测各组小鼠学习、记忆能力;Western blot检测各组小鼠海马ULK1、P62、LC3Ⅱ/LC3Ⅰ的蛋白表达;免疫组织化学方法检测mTOR的表达;ELISA法检测Aβ1-42水平。结果与NT组比较,Tg组小鼠寻找平台的逃避潜伏期延长及穿越目的象限的次数减少(P<0.05),海马ULK1表达降低、P62、LC3Ⅱ/LC3Ⅰ、Aβ1-42表达升高(P<0.05),EGCG治疗组较Tg组各异常指标均明显改善(P<0.05)。结论EGCG可以改善APP/PSl小鼠空间学习记忆能力,其机制可能与提高海马神经元自噬活性,减少脑内Aβ沉积有关。     

Cerebral metabolism of [3H]-p-chloroamphetamine

The time-dependent metabolism of intraventricularly administered $\text{[}{}^3\text{H}\text{]-p-}\text{chloroamphetamine}$ was followed. The parent compound and its metabolites were recovered by high pressure liquid chromatography and characterized by high pressure liquid chromatography, thin-layer chromatography, and gas chromatography-mass spectrometry. By 4 hr after injection, two major toluene-soluble metabolites were present in brain. Their biological half-lives were different from the parent compound. On the basis of their analyses, one of the metabolites is p-chloronorephedrine, the other (P₃) is as yet unidentified. Pretreatment with Lilly 110140 prevented or markedly reduced the synthesis of both p-chloronorephedrine and P₃. Iprindole prevented the synthesis of p-chloronorephedrine. The P₃ appeared first in the brain then in the liver, suggesting that both of these organs can metabolize p-chloroamphetamine to this compound. The metabolites were recovered primarily from the nuclear and microsomal fractions following subcellular fractionation of the brain, with small quantities present in the synaptosomal fraction. The level of metabolites was higher in the brainstem than in the neocortex. Glutathione, administered simultaneously with p-chloroamphetamine either intraventricularly or intraperitoneally failed to alter the toxicity of p-chloroamphetamine.     

Clevudine University of Georgia/Abbott/Bukwang/Triangle/Yale University

The pyrimidine analog, clevudine (L-FMAU: 2'-fluoro-5-methyl-beta-L-arabinofuranosyluridine) is a potent antihepatitis B virus (HBV) and anti-Epstein-Barr virus (EBV) agent, discovered by researchers at the University of Georgia, in collaboration with Yale University and Bukwang. Bukwang transferred its technology to Triangle Pharmaceuticals in 1998 together with a license to develop clevudine worldwide except Korea [279649], [281942]. In June 1999, Triangle and Abbott Laboratories entered into a strategic alliance to copromote antiviral products including L-FMAU [326798]. In September 2000, Triangle Pharmaceuticals Inc initiated a 30-day phase I/II evaluation of clevudine in HBV-infected patients [381755]. Clevudine is a much less toxic derivative of the toxic agent P-D-FMAU. The mechanism of action of clevudine is not yet clear, but the agent induces a rapid decrease in HBV nucleic acid as doses increase from 0.3 to 10 mg/kg [319145]. It is believed that the target for clevudine lies in the viral replication mechanism. Clevudine is phosphorylated to the triphosphate form intracellularly. This is removed slowly from the cells, thus exerting a sustained inhibitory antiviral activity [178173], [320720], [320721].     

Spotlight from the American Society for Preventive Cardiology on Key Features of the 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guidelines on the Management of Blood Cholesterol

The 2018 AHA/ACC/AACVPR/AAPA/ABC/ACPM/ADA/AGS/APhA/ASPC/NLA/PCNA Guideline on the Management of Blood Cholesterol retains focus on recommendations for statin treatment in the original four statin-eligible groups [those with atherosclerotic cardiovascular disease (ASCVD), diabetes, low-density lipoprotein cholesterol (LDL-C) ≥ 190 mg/dL, and higher risk primary prevention] without the use of treatment initiation or target LDL-C levels from the earlier 2013 American College of Cardiology/American Heart Association (ACC/AHA) guideline, but has several new features. First, patients with primary prevention are divided into those who are at low (< 5%), borderline (5% to < 7.5%), intermediate (7.5% to < 20%), and high (≥ 20%) risk based on the ASCVD risk estimator. Moreover, the new guideline goes further to consider a wider range of factors [now called “risk enhancers”—premature family history of ASCVD, persistently high LDL-C, chronic kidney disease (CKD), metabolic syndrome, conditions specific to women, inflammatory diseases, and high-risk ethnicities] that can be used to better inform the treatment decision. Moreover, more detailed recommendations on how the results of coronary calcium scanning can be used to inform the treatment decision are provided, including how it may be used to “de-risk” certain patients for delaying or avoiding the use of statin therapy. There are also specific sections for cholesterol management in other patient subgroups including women, children, certain ethnic groups, those with CKD, chronic inflammatory disorders and HIV, as well as discussion on the management of hypertriglyceridemia. Importantly, for persons with known ASCVD, a distinction is made for those who are at “very high risk” based on having had two major ASCVD events or one major event and two or more other high risk conditions, such as diabetes or other major risk factors, or bypass surgery or percutaneous intervention. Finally, the concept of a threshold LDL-C for initiating a non-statin therapy (after considering highest tolerated statin dosage) is provided, with ezetimibe recommended as the key non-statin to be added if the LDL-C still remains ≥ 70 mg/dL for all ASCVD patients, and in those who are at “very high risk”, further consideration for using a proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. While the new guideline does have greater detail (and arguably, complexity), the refinements provide a strategy for guiding the clinician to target both statin and non-statin therapy to those most likely to derive benefit.     

Pitavastatin Nissan/Kowa Yakuhin/Novartis/Sankyo

Pitavastatin (nisvastatin) is an HMG CoA reductase inhibitor being developed jointly by Nissan, Kowa Kogyo, Novartis and Sankyo for the potential treatment of atherosclerosis and hyperlipidemia.     

Amlodipine/Valsartan/Hydrochlorothiazide

Amlodipine/valsartan/hydrochlorothiazide (HCTZ) is a fixed-dose combination of the well established antihypertensive agents amlodipine (a calcium channel antagonist), valsartan (an angiotensin II receptor antagonist), and HCTZ (a thiazide diuretic). In patients with moderate or severe hypertension, triple combination therapy with amlodipine + valsartan + HCTZ produced significantly greater reductions from baseline in mean sitting systolic and diastolic BP (msSBP and msDBP) than either valsartan + HCTZ, amlodipine + HCTZ, or amlodipine + valsartan in a large, 8-week, randomized, double-blind, multinational, phase III trial. Furthermore, the proportion of patients achieving overall BP control at endpoint was significantly greater with the triple combination regimen than with any of the dual regimens, with significantly more triple combination recipients achieving msSBP and msDBP control at each assessment throughout the trial. Subgroup analyses of this study suggested that amlodipine + valsartan + HCTZ was generally more effective in reducing BP and providing overall BP control than the dual combination therapies, irrespective of age, race, gender, ethnicity, or hypertension severity. Several smaller studies provide data that support the efficacy of amlodipine + valsartan + HCTZ in patients whose BP is inadequately controlled with amlodipine + valsartan, amlodipine + HCTZ, or valsartan + HCTZ dual combination therapy. Treatment with amlodipine + valsartan + HCTZ for up to 8 weeks was generally well tolerated in the large, phase III trial, with most adverse events being transient and of mild to moderate severity.     

N-Nitroso-N-Methyldodecylamine and N-Nitroso-N-Methyltetradecylamine in household dishwashing liquids

Eleven household dishwashing liquids and four household surface cleaners were analysed for N-nitroso-N-methyldodecylamine and N-nitroso-N-methyltetradecylamine by gas chromatography with detection using a Thermal Energy Analyzer. Both nitrosamines were found in three of the dishwashing detergents and one of the surface cleaners. [1-¹⁴C]-N-Nitroso-N-methyldodecylamine was used to determine recoveries, which were between 65 and 88%. Levels of N-nitroso-N-methyldodecylamine ranged from 112 to 661 ppb and those of N-nitroso-N-methyltetradecylamine from 46 to 151 ppb. A simple method was developed to screen the products for N,N-dimethyldodecylamine-N-oxide, a surfactant ingredient suspected of being the source of these nitrosamines. By application of this method it was established that all of the products formulated with this amine oxide contained these two nitrosamines, whereas in products that did not contain this ingredient, these nitrosamines were not detected.     

PROTON AND POTASSIUM TRANSPORT BY H+/K+-ATPases

1. H⁺/K⁺-ATPases are members of the P-type ATPase multigene family. The prototypical H⁺/K⁺-ATPase is the protein that acidifies gastric luminal contents. The physiological and pharmacological significance of this pump has led to a detailed investigation of its biochemistry and molecular and cell biology. 2. Recently, a number of closely related H⁺/K⁺-ATPase isoforms have been discovered. These isoforms are present in organs other than the stomach, including the colon and kidney, where they contribute to acid—base and potassium homeostasis. The structure, expression and physiological roles of the gastric H⁺/K⁺-ATPase and other isoforms are reviewed.     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

EFFECTS OF THE OPIOID PEPTIDES [MET5]ENKEPHALIN-ARG6-PHE7 AND [MET5]ENKEPHALIN-ARG6-GLY7-LEU8 ON CHOLINERGIC NEUROTRANSMISSION IN THE RABBIT ISOLATED ATRIA

1. The effect of the opioid peptides [Met5]enkephalin-Arg6-Phe7 (MEAP) and [Met5]enkephalin-Arg6-Gly7-Leu8 (MEAGL) were compared with those of [Leu5]enkephalin and [D-Ala2,Met5]enkephalinamide (DAME) on cholinergic neurotransmission in the rabbit isolated atria. 2. Rabbit isolated atria had a resting rate of 190 beats/min. In the presence of the beta-adrenoceptor antagonist propranolol (0.3 mumol/l), atria responded to electrical field stimulation with a cholinergically mediated negative chronotropic response. The opioid peptides had no effect on the resting rate, but inhibited the negative chronotropic response to field stimulation. The IC50 values for inhibiting the cholinergic responses were 1.4 mumol/l for [Leu5]enkephalin (LE), 1.4 mumol/l for MEAP, 1.3 mumol/l for MEAGL and 0.2 mumol/l for DAME. Responses of a similar magnitude to exogenous acetylcholine were unaffected. 3. Thus, MEAP, MEAGL and LE had similar potencies but DAME was about seven times more potent in inhibiting cholinergic neurotransmission in the rabbit isolated atria. The site of inhibition appears to be prejunctional.