内洋地黄素拮抗剂上调缺血再灌注损伤大鼠心肌细胞膜钠泵亚基的表达

目的： 观察内洋地黄素特异性拮抗剂地高辛抗血清对心肌缺血再灌注（MIR）损伤大鼠心肌组织内洋地黄素水平、钠泵活性、线粒体总钙浓度以及钠泵各亚基基因表达的影响，探讨内洋地黄素在心肌缺血再灌注损伤中的作用及其机制。方法: 将56只雄性SD大鼠随机分成7组，每组8只。假手术对照组（sham）：丝线穿过左冠状动脉前降支，但不结扎；缺血再灌注组（MIR）：结扎左冠状动脉前降支30 min，再灌注45 min；生理盐水组（NS）、维拉帕米组（Ver）、小剂量、中剂量、大剂量地高辛抗血清组（ADA）：于再灌注前5 min经股静脉分别注射生理盐水、维拉帕米5 mg·kg^-1、地高辛抗血清8.6 mg·kg^-1、 17.3 mg·kg^-1、34.5 mg·kg^-1，容积均为5 mL·kg^-1，5 min内注射完毕，其余同MIR模型组。再灌注结束后，立即取缺血区左室心肌检测心肌匀浆内洋地黄素水平、心肌细胞膜Na+ K+_ATP酶和Ca2+_Mg2+_ATP酶活性、线粒体总钙浓度；分别采用RT-PCR及Western blotting方法和免疫组化方法检测心肌钠泵α₁、α₂、α₃和β₁亚基mRNA及蛋白水平基因表达的改变。结果: 心肌缺血再灌注损伤时，心肌组织内洋地黄素水平明显升高，心肌细胞膜钠泵和Ca2+_Mg2+_ATP酶活性显著下降，线粒体总钙浓度升高，钠泵α₁、α₂、α₃和β₁亚基在mRNA及蛋白水平基因表达均明显下降；维拉帕米除具有降低线粒体总钙浓度外，对其它各项指标无明显影响。地高辛抗血清呈剂量依赖性地显著降低心肌组织内洋地黄素水平，恢复细胞膜钠泵和Ca2+_Mg2+_ATP酶活性，降低线粒体总钙浓度，上调钠泵α₁、α₂、α₃和β₁亚基mRNA及蛋白水平的基因表达。结论: 心肌缺血再灌注促进机体内洋地黄素分泌增加，后者通过下调心肌细胞膜上的钠泵α₁、α₂、α₃和β₁亚基基因表达抑制钠泵活性，进而抑制Ca2+_Mg2+_ATP酶活性，导致线粒体内钙超载，介导心肌缺血再灌注损伤。内洋地黄素特异性拮抗剂地高辛抗血清通过阻断内洋地黄素的生物学作用，上调钠泵各亚基的基因表达，发挥其抗心肌缺血再灌注损伤的作用。     

高渗葡萄糖处理后脱水红细胞膜Na^＋—K^＋—ATP酶的变化

目的体外观测高渗葡萄糖处理后脱水红细胞膜Na+-K+-ATP酶的变化.方法对全血与50%葡萄糖(50%GS)溶液混合前后红细胞膜Na+-K+-ATP酶的变化,以及脱水红细胞于等渗条件下其Na+-K+-ATP酶的恢复情况进行了测定.结果(1)全血与50%GS溶液混合后其脱水红细胞膜Na+-K+-ATP酶活性比混合前显著降低(0.307μmol/mg.h-1±0.073μmol/rmg.h-1vs.0.396μmol/mg.h-1±0.104μmol/mg.h-1,P＜0.05).(2)等渗条件下脱水红细胞膜Na+-K+-ATP酶的活性由异常逐步恢复至正常(即刻0.349μrrol/mg.h-1±0.0817μmol/mg.h-1;2h0.355μmol/mgh-1±0.0946μmol/m]g.h-1,4h038μmol/mg.h-1±0.0877μmol/mg.h-1,P＜0.01).结论高渗葡萄糖处理后脱水红细胞膜Na+-K+-ATP酶活性显著降低,但其异常变化在等渗条件下是可逆的.     

参麦注射液对大鼠急性心肌缺血再灌注损伤的影响

目的通过在体实验初步探讨参麦注射液对大鼠急性心肌缺血再灌注损伤的防治作用及相关机制.方法通过结扎左冠状动脉前降支10min,松开丝线复灌15min,来复制大鼠急性心肌缺血再灌注损伤的模型.结扎的同时经股静脉缓慢推注参麦注射液,观察参麦注射液对再灌注性心律失常的影响,并测定心肌组织匀浆中MDA的含量、SOD的活力、Na+,K+-ATP酶和Ca2+-ATP酶的活性.此外,还通过光镜和电镜来观察参麦注射液对再灌后心肌超微结构的影响.结果(1)模型组大鼠再灌注性心律失常的发生率为88.9%,持续时间为(142.8±73.4)s,参麦注射液可以使再灌注性心律失常的发生率降至33.3%,持续时间缩短至(31.7±20.1)s,两组之间有明显差异(P＜0.05).(2)模型组心肌组织匀浆SOD的活力为(27.287±3.449)×103NU/gprotein,参麦注射液治疗后SOD活力升高至(30.791±1.676)×103NU/gprotein,二者之间有显著差异(P＜0.05);模型组MDA的含量为(0.319±0.0515)μmol/gprotein,参麦注射液治疗组为(0.262±0.0472)μmol/gprotein,MDA的含量明显降低(P＜0.05).(3)模型组Na+,K+-ATP酶的活性为(0.3420±0.0391)mmolPi*g-1protein*h-1,参麦注射液治疗后其活性升高至(0.3950±0.0265)mmolPi*g-1protein*h-1,二者之间有显著差异(P＜0.01);模型组Ca2+-ATP酶的活性为(0.3450±0.0438)mmolPi*g-1protein*h-1,参麦注射液治疗组为(0.4270±0.0624)mmolPi*g-1protein*h-1,其活性明显升高(P＜0.01).(4)正常组和假手术组光镜显示心肌细胞排列整齐,横纹清晰,结构正常,电镜显示肌原纤维清晰,排列整齐,线粒体无肿胀,嵴密集,排列规则.模型组光镜显示心肌细胞排列紊乱,横纹不清晰,胞浆嗜酸性变,有明显的空泡变性,间质严重水肿,毛细血管内有红细胞淤积,电镜显示肌原纤维模糊不清,线粒体肿胀明显,嵴稀疏,排列紊乱.经参麦注射液治疗后,光镜显示心肌细胞排列整齐,横纹清晰,形态接近正常,电镜显示肌原纤维清晰,排列基本整齐,线粒体肿胀减轻,嵴排列也较规则.结论参麦注射液能降低再灌注性心律失常的发生率,提高心肌组织中SOD的活力,降低MDA的含量,提高Na+,K+-ATP酶和Ca2+-ATP酶的活性,对缺血再灌注损伤引起的心肌超微结构的损伤有明显的保护作用.参麦注射液防治心肌缺血再灌注损伤的机制与增强机体清除氧自由基的能力,减轻脂质过氧化反应,拮抗自由基的毒性作用,维持离子泵的正常转运,减轻钙超载,从而维持了心肌细胞膜和线粒体的正常功能有关.     

体外反搏对局部血管紧张素原与肾素基因表达的影响

目的探讨局部血管紧张素原(angiotensinogen,ATG)与肾素在增强型体外反搏(enhancedexternalcounterpulsation,EECP)治疗心肌缺血时的改变。方法利用冠状动脉结扎法造成犬急性心肌缺血动物模型。观察EECP治疗时缺血心肌、肾脏、肺脏和主动脉处肾素活性的改变。应用反转录-聚合酶链式反应(RT-PCR)观察组织中ATG与肾素mRNA的表达。结果反搏组缺血心肌肾素活性明显低于缺血组[(2.36±0.59)ng·ml-1·h-1对(3.21±0.67)ng·ml-1·h-1,P<0.05]。反搏组缺血心肌中ATG、肾素以及主动脉肾素mRNA也明显低于缺血组(0.56±0.16对0.71±0.15,1.12±0.16对1.37±0.22,P<0.05;0.96±0.11对1.18±0.18,P<0.05)。结论EECP能通过抑制心血管肾素mRNA的表达来抑制肾素活性,同时对ATGmRNA有抑制作用。这可能是EECP抑制心血管局部血管紧张素(Ang)Ⅱ水平,从而保护缺血心肌的机制之一。     

不同液体复苏方法对孕兔失血性休克各脏器ATP酶活性的影响

目的： 探讨限制性液体复苏对非控制性失血性休克孕兔腹腔内失血量、补液量、血细胞比容（Hct）及各脏器Na＋-K＋-ATP酶活性和Ca2＋-Mg2+-ATP酶活性的改变,以分析其对休克后近期能量代谢的影响。方法: 将30只孕中晚期新西兰大白兔随机分为5组：(1)假休克组（SS）;(2)休克未处理组（SH）;(3)传统复苏组（NS）;(4)生理盐水限制性输液复苏组（NH）;(5)高渗高胶液体限制性复苏组（HHH）。实验设计分为休克期（T0-T30 min）、休克复苏期T30（T30-T90 min）及休克复苏期T180（T90-T180 min）。休克期：除SS组外各组动物均接受离断子宫系膜小动脉及颈动脉放血至平均动脉压（MAP）40-45 mmHg,制作非控制性失血性休克模型。休克复苏期T30：NS组接受生理盐水（4 mL/kg）及林格氏液快速输注以维持MAP在80 mmHg左右;NH、HHH组接受生理盐水（4 mL/kg）或高渗高胶液（6％羟乙基淀粉＋7.5％氯化钠）（4 mL/kg）及林格氏液输注以维持MAP在60 mmHg左右。休克复苏期T180：除SS组外各组动物均接受手术止血及输血输液治疗。实验中监测血细胞比容(Hct),统计腹腔内失血量、补液量。实验结束后处死动物取心脏、肺脏、肝脏、肾脏、小肠和骨骼肌组织检测Na＋-K＋-ATP酶、Ca2＋-Mg2+-ATP酶活性。 结果: 腹腔内失血量与补液量：NH［(4.3±0.2) mL/kg,(47.2±4.1) mL/kg］与HHH［(4.1±0.3) mL/kg,(44.9±4.3) mL/kg］均明显低于NS［(5.5±0.2) mL/kg,(65.5±3.8) mL/kg］。T180 min：Hct在NH(21.0%±2.1%)与HHH(21.5%±1.8%)均显著高于NS(14.2%±1.5%)及SH (12.5%±1.4%)。失血性休克引起Na＋-K＋-ATP酶、Ca2＋-Mg2+-ATP酶活性的升高;其中骨胳肌、心肌、肝、肾组织Na＋-K＋-ATP酶活力在NH（5.42±1.41,4.54±2.01,4.13±0.62,3.42±0.84）与HHH（3.97±0.91,2.94±0.66,3.22±1.42,3.03±0.53）均显著低于NS（7.34±1.41,6.23±1.53,6.11±0.97,5.82±0.69）与SH（9.11±0.52,8.40±1.08,7.04±1.13,6.55±1.45）;肺及肠组织中,NH、HHH、NS组比较无显著差异。结论: 孕兔非控制性失血性休克后导致能量代谢障碍,不同器官影响不同,其中心肌和骨胳肌ATP酶活性改变显著。传统复苏加剧了能量代谢的紊乱,限制性复苏相对稳定了能量代谢,减轻对细胞生物膜及细胞器的损害。     

高胆固醇血症对大鼠心室肌细胞离子电流的作用

目的：观察高胆固醇血症对大鼠心室肌细胞离子电流的作用。方法： 通过全细胞膜片钳技术记录用酶解法分离的正常和高胆固醇饮食的大鼠心室肌细胞离子电流。结果： 高胆固醇组（组Ⅱ）血清总胆固醇水平明显高于正常组（组Ⅰ）［（3.10±0.62)mmol·L-1 vs (1.18±0.37)mmol·L-1, P<0.01, n=20］。组Ⅱ血清甘油三酯也明显高于组Ⅰ［（1.51±0.30)mmol·L-1 vs (0.43±0.15)mmol·L-1, P<0.01, n=20］。组Ⅱ大鼠心室肌细胞动作电位时程（APD）与组I相比明显延长，APD50从(70.86±8.12)ms延长至(116.16±6.90)ms (n=10, P<0.01); APD90 从(95.10±7.27)ms延长至(144.04±7.39)ms (n=10, P<0.01)；在实验电压 -120 mV， Ik1从(-16.98±4.54) pA/pF（组I）增加到(-19.92±4.08) pA/pF（组Ⅱ）(n=12, P<0.05)；在实验电压 0 mV， ICa-L从(-8.56±1.29) pA/pF（组Ⅰ）减少到(-5.24±0.90) pA/pF（组Ⅱ）(n=10, P<0.01)；在实验电压 +60 mV，Ito从(13.20±1.97) pA/pF（组I）减少到(10.30±1.97) pA/pF（组Ⅱ）(n=8, P<0.05)。结论： 高胆固醇血症可显著改变心肌细胞离子电流密度的大小，对心脏具有毒性作用。     

抑制核因子-κB活性对TNF-α诱导的肾小球系膜细胞血管紧张素原表达的影响

目的： 研究抑制核因子-κB（NF-κB）活性对肿瘤坏死因子-α（TNF-α）诱导的SD大鼠肾小球系膜细胞血管紧张素原表达及血管紧张素Ⅱ产生的影响。方法：分离SD大鼠肾小球系膜细胞分为下列3组：对照组,TNF-α处理组,TNF-α＋NF-κB抑制剂吡咯烷二硫氨基甲酸酯（PDTC）处理组（TNF-α＋PDTC处理组）。以电泳迁移率变动分析法（electrophoretic mobility shift assay,EMSA）检测NF-κB 活性,放射免疫分析法检测培养液血管紧张素Ⅱ水平,RT-PCR方法检测血管紧张素原mRNA表达,蛋白质印迹法（Western blotting）检测血管紧张素原蛋白表达。结果： TNF-α处理组NF-κB活性［（20.67±9.14）×102 μg/cell］显著高于对照组［（8.25±4.35）×102 μg/cell,P<0.01］与TNF-α＋PDTC处理组［（7.20±4.57）×102 μg/cell,P<0.01］,TNF-α＋PDTC处理组与对照组比较无显著差异。血管紧张素原mRNA表达TNF-α处理组（0.27±0.05）显著高于对照组（0.20±0.05,P<0.05）,与TNF-α＋PDTC处理组（0.22±0.06,P>0.05）比较无显著差异;蛋白表达TNF-α处理组［（0.60±0.19） μg/cell］显著高于对照组［（0.37±0.15） μg/cell,P<0.05］及TNF-α＋PDTC处理组［（0.37±0.17） μg/cell,P<0.05］,TNF-α＋PDTC处理组与对照组比较无显著差异（P＞0.05）。培养液血管紧张素Ⅱ水平在TNF-α处理组［（9.73±2.38）×10-5 ng·L-1/cell］显著高于对照组［（7.50±1.51）×10-5ng·L-1/cell,P<0.05］及TNF-α＋PDTC处理组［（6.94±1.46）×10-5 ng·L-1/cell,P<0.05］,TNF-α＋PDTC处理组与对照组比较无显著差异（P＞0.05）。结论：TNF-α可激活SD大鼠肾小球系膜细胞NF-κB,并诱导血管紧张素原表达及血管紧张素Ⅱ产生增加;抑制NF-κB活性可降低血管紧张素原表达及血管紧张素Ⅱ产生。结果提示NF-κB介导TNF-α诱导的肾小球系膜细胞血管紧张素原表达及血管紧张素Ⅱ产生。     

EETs下调在高脂诱导肥胖相关高血压中的作用

目的：探讨肾脏环氧二十碳烯酸（epoxyeicosatrienoic acids，EETs)在高脂饮食诱导的幼年型肥胖相关高血压发病机制中的作用。方法:以高脂饮食喂养3周龄SD雄性大鼠至12周龄，监测正常饮食与高脂饮食两组大鼠的体重与血压的变化，并以Western blotting和反相HPLC方法比较两组大鼠的的肾脏各部分EETs活性的差异。结果:高脂饮食大鼠体重在第8周龄，收缩压在第11周龄，均明显高于正常饮食组［(328±23）g vs (273±21)g, (153.0±8.6)mmHg vs (134.0±7.7)mmHg, P<0.05］,而两组肾微小血管产生EETs的活性无差异；高脂饮食组肾皮质和乳头部产生EETs的活性低于正常饮食组［（75.4±9.2)nmol·g-1·min-1 vs (138.1±10.3）nmol·g-1·min-1，（55.8±6.2)nmol·g-1·min-1 vs (121.6±11.3）nmol·g-1·min-1，P<0.05］，同时Western blotting表明产生EETs的细胞色素P450(CYP)表达也出现相应的降低。结论:高脂饮食诱导的幼年型肥胖相关高血压的发生可能与肾皮质和乳头部的EETs下调有关。     

地塞米松预处理减轻大鼠再灌注性心律失常的实验研究

目的： 探讨地塞米松预处理对大鼠再灌注性心律失常的作用及机制。方法: SD大鼠随机分成地塞米松组、对照组，分别予地塞米松和生理盐水预处理。预处理后构建缺血再灌注损伤动物模型，观察再灌注期间心律失常的发生；Western blotting法和免疫组化法观察心肌HSP72表达变化；测定心肌MDA、SOD、CAT、GSH-Px水平及心肌细胞膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性。结果: 与对照组相比，地塞米松组室性心律失常的积分减少（P<0.01）、持续时间缩短（P<0.05）；HSP72的表达增加（P<0.05）；MDA降低（P<0.01），SOD、CAT、GSH-Px均升高（P<0.05）；Na+-K+-ATP酶增加（P<0.01），Ca2+-Mg2+-ATP酶无明显变化（P>0.05）。结论: 地塞米松预处理减少再灌注室性心律失常，其机制可能与其上调HSP72、Na+-K+-ATP酶、抗氧化酶的表达及抑制脂质过氧化反应有关。     

耗竭心肌细胞内糖原对缺血后再灌注心肌的保护作用及其机制

本工作在离体大鼠等容收缩心脏模型上观察耗竭心脏细胞内糖原对心肌缺血再灌注损伤的影响。心脏富氧灌流30 min后,随机分为三组:Ⅰ、富氧组:富氧灌流75min。Ⅱ、对照组:常规K-H液富氧灌流15min,旷置30min,再灌流30min。Ⅲ、耗竭糖原组:先用充N_2(95%N_2:5%CO_2)的K-H液灌流15min,余同组Ⅱ。结果表明。耗竭心肌细胞内糖原可提高再灌注后心脏血液动力学的恢复;冠脉流出液中LDH活性及心肌组织MDA含量降低,线粒体及胞浆液中GSH-Px有较高的活性;心肌组织Na~+,Ca~(2+)超负荷减轻。说明耗竭心肌细胞内糖原可通过减少细胞内H~+的生成抑制Na~+/H~+交换,从而明显减轻心肌缺血再灌注损伤。     

野生型和突变型GPⅢa真核表达载体的构建与鉴定

目的 构建人野生型和突变型血小板糖蛋白Ⅲa(GPⅢa)真核表达重组质粒PeDNA3.1(+)Ⅲa和PeDNA3.1(+)ⅢanT1565C.方法 选择人红白血病细胞提取人基因组总RNA,经过逆转录-聚合酶链反应后,采用定点诱变技术,通过三轮聚合酶链式反应将第2外显子1565位基因由T置换为C,把最终产物与质粒peDNA...     

β-淀粉样蛋白对D-半乳糖致衰大鼠海马线粒体膜流动性的影响

目的:研究β-淀粉样蛋白(β-AP)对衰老大鼠海马线粒体膜流动性的影响。方法:用D-半乳糖(D-gal)建立衰老动物模型,并且海马内微注射β-AP;检测各组大鼠学习记忆行为;以DPH为荧光探针,测定大鼠海马线粒体膜粘滞系数,同时测定海马Na⁺-K⁺ATP酶活性的变化。结果:D+A组大鼠Na⁺-K⁺ATP酶活性明显低于D组和A组(P＜0.05),与N组比较有显著差异(P＜0.01);海马线粒体膜流动性显著低于N组(P＜0.01),与D组和A组比较也有显著差异(P＜0.05)。结论:β-AP与D-gal联合可导致脑海马自由基损伤效应加重,海马线粒体膜流动性显著降低,Na⁺-K⁺ATP酶活性明显降低。     

Safety and Pharmacokinetic Profile of Multiple Escalating Doses of (+)-Calanolide A,a Naturally Occurring Nonnucleoside Reverse Transcriptase Inhibitor,in Healthy HIV-Negative Volunteers

Abstract

Background: (+)-Calanolide A is a naturally occurring nonnucleoside reverse transciptase inhibitor (NNRTI) that exhibits enhanced activity against HIV-1 isolates with the Y181C mutation and retains activity against HIV-1 isolates with dual Y181C and K103N mutations. Previous studies have demonstrated that (+)-calanolide A has a favorable safety profile in both animal and human subjects. Method: In this study, the safety and pharmacokinetics of multiple escalating doses of (+)-calanolide A were evaluated in a total of 47 healthy, HIV-seronegative individuals. Results: All adverse events seen in the study were mild to moderate in intensity and were transient. The most common adverse events seen were headache, dizziness, nausea, and taste perversion (oily aftertaste). Laboratory abnormalities were determined to be clinically insignificant or unrelated to (+)-calanolide A administration. No dose-related pattern in adverse event or laboratory abnormality incidence was apparent. In all cohorts examined, administration of (+)-calanolide A produced highly variable plasma levels and absorption profiles. No accumulation of parent compound was seen over the 5-day treatment course, with the day 5 area under the curve (AUC) being approximately one half of that seen on the first day of dosing. Steady-state trough plasma levels were determined in the two highest dose cohorts (600 mg and 800 mg bid for 5 days). Mean elimination half-life in the two highest dosing cohorts combined was 15.5 hours in men and 35.2 hours in women. Conclusion: These pharmacokinetic properties, together with the benign safety profile, and unique in vitro resistance pattern warrant the continued development of this potential new antiviral agent.     

高胆红素血症大鼠脑干听觉诱发电位与脑组织NO含量及Na+-K+ATP酶活性的变化

目的： 探讨脑干听觉诱发电位(BAEP)在早期监测高胆红素血症听力和脑损伤中的作用及脑组织一氧化氮(NO)与胆红素诱导的听力和脑损伤的关系。方法: 15 d SD大鼠腹腔注射不同剂量(30 mg/kg、60 mg/kg、90 mg/kg、120 mg/kg和150 mg/kg)胆红素溶液以制备高胆红素血症动物模型，微量胆红素测定仪测定血清胆红素浓度，重氮法测定脑组织胆红素浓度，定磷法测定脑组织中Na+-K+ATP酶活性，硝酸酶还原法测定脑组织NO含量，诱发电位仪检测BAEP。结果: 建模后高剂量组（120 mg/kg和150 mg/kg）部分大鼠出现异常神经行为活动；建模6 h后，除低剂量（30 mg/kg）组外，各实验组大鼠血清和脑组织胆红素浓度及脑组织NO含量显著升高，脑组织Na+-K+ATP酶活性显著降低，BAEP的波峰潜伏期(PL)和波峰间潜伏期(IPL)显著延长；且BAEP的PL与IPL和脑组织NO含量与Na+-K+ATP酶活性的变化均与脑组织胆红素水平显著相关。结论: BAEP的PL和IPL是早期监测高胆红素血症听力和脑损伤的无创性指标，NO的过量产生可能参与了胆红素诱导的听力和脑损伤的发病过程。     

Regioselective Synthesis and Structures of (+)‐Catechin‐Aldehyde Polycondensates

The polymerization of (+)‐catechin with various aldehydes has been performed in the presence of an acid catalyst to produce catechin‐aldehyde polycondensates in high yields. NMR analysis of the product showed that the polymerization regioselectively proceeded to form the polymer linked by an ethyl bridge through C‐6 and C‐8 positions of catechin A ring. Specific rotation and CD measurements of the polymer showed the change of chemical bonding and conformation at near chiral center. The direction of specific rotation was changed from dextrorotatory (+) to levorotatory (?) and a new chiral center was formed on the bridge moiety between the catechin units. Computer simulation suggests the coiled interactions in the polymer by hydrogen bonds between hydroxyl groups and pyranic oxygens.

     

Subject Index to Volume 21

ABSTRACT

A novel amperometric immunosensor, based on graphite paste (graphite powder and paraffin oil), has been constructed for the assay of l-T₄. The graphite paste is impregnated with mouse monoclonal anti-(+)-3,3′,5,5′-tetraiodo-l-thyronine (anti-l-T₄). The immunosensor can be reliably used for the assay of l-T₄ in thyroid and in drugs, using chronoamperometry technique, at ppt up to ppb concentration levels. The potential used for l-T₄ assay was +450 mV vs. Ag/AgCl electrode. The surface of the immunosensor can be regenerated by simply polishing, obtaining fresh immunocomposite ready to be used in a new assay.     

IL-17 and related cytokines involved in systemic sclerosis: Perspectives

Systemic sclerosis (SSc) is a multisystemic, complex, and rare disease of connective tissue, with high morbidity and mortality, and without specific treatment. The disease is characterized by three main principles: vascular disease, autoantibody production and inflammation, and fibrosis. Since it is well defined that SSc is characterized by elevated production of TGF-β, IL-6, and IL-1, all of them cytokines related to Th17 differentiation, the hypothesis is that this disease may be strongly related to a polarization of the immune response towards the Th17 pathway. Considering the importance of a better understanding of the pathophysiology of Th17 pathway in SSc, this article aims to propose an update for a better understanding of current knowledge on main cytokines secreted by the Th17 cells (IL-17?A, IL-21, and IL-22) and the future prospects in the current disease.     

Evidence that intrathecal morphine-3-glucuronide may cause pain enhancement via toll-like receptor 4/MD-2 and interleukin-1β

Morphine-3-glucoronide (M3G) is a major morphine metabolite detected in cerebrospinal fluid of humans receiving systemic morphine. M3G has little-to-no affinity for opioid receptors and induces pain by unknown mechanisms. The pain-enhancing effects of M3G have been proposed to significantly and progressively oppose morphine analgesia as metabolism ensues. We have recently documented that morphine activates toll-like receptor 4 (TLR4), beyond its classical actions on μ-opioid receptors. This suggests that M3G may similarly activate TLR4. This activation could provide a novel mechanism for M3G-mediated pain enhancement, as (a) TLR4 is predominantly expressed by microglia in spinal cord and (b) TLR4 activation releases pain-enhancing substances, including interleukin-1 (IL-1). We present in vitro evidence that M3G activates TLR4, an effect blocked by TLR4 inhibitors, and that M3G activates microglia to produce IL-1. In vivo, intrathecal M3G (0.75 μg) induced potent allodynia and hyperalgesia, blocked or reversed by interleukin-1 receptor antagonist, minocycline (microglial inhibitor), and (+)-and (−)-naloxone. This latter study extends our prior demonstrations that TLR4 signaling is inhibited by naloxone nonstereoselectively. These results with (+)-and (−)-naloxone also demonstrate that the effects cannot be accounted for by actions at classical, stereoselective opioid receptors. Hyperalgesia (allodynia was not tested) and in vitro M3G-induced TLR4 signaling were both blocked by 17-DMAG, an inhibitor of heat shock protein 90 (HSP90) that can contribute to TLR4 signaling. Providing further evidence of proinflammatory activation, M3G upregulated TLR4 and CD11b (microglial/macrophage activation marker) mRNAs in dorsal spinal cord as well as IL-1 protein in the lumbosacral cerebrospinal fluid. Finally, in silico and in vivo data support that the glucuronic acid moiety is capable of inducing TLR4/MD-2 activation and enhanced pain. These data provide the first evidence for a TLR4 and IL-1 mediated component to M3G-induced effects, likely of at least microglial origin.     

人β2-微球蛋白在pET-22b(+)表达载体中的克隆、表达与纯化

目的：构建人β2-microglobulin(β2-M)基因的原核表达载体，在大肠杆菌中高效表达β2-M蛋白质并进行纯化，为构建MHC-肽四聚体奠定基础。方法：从人外周血单核细胞中提取总RNA，采用RT-PCR技术扩增出去除信号肽部分的β2-M基因，克隆人pET-22b( )表达载体进行诱导表达。采用离子交换层析和凝胶过滤方法对表达产物进行纯化，用ELISA和Westem blot法进行免疫学鉴定。结果：成功地构建了表达载体pET-22b( )-β2-M，对表达产物进行了纯化，并对表达产物进行了鉴定。结论：获得β2-M蛋白的高效表达，为进一步利用亲和素在体外构建MHC-Ⅰ类分子肽四聚体奠定了基础。     

Peri-pubertal maturation after developmental disturbance: a model for psychosis onset in the rat

Schizophrenia is thought to be associated with abnormalities during neurodevelopment although those disturbances usually remain silent until puberty; suggesting that postnatal brain maturation precipitates the emergence of psychosis. In an attempt to model neurodevelopmental defects in the rat, brain cellular proliferation was briefly interrupted with methylazoxymethanol (MAM) during late gestation at embryonic day 17 (E17). The litters were explored at pre- and post-puberty and compared with E17 saline-injected rats. We measured spontaneous and provoked locomotion, working memory test, social interaction, and prepulse inhibition (PPI). As compared with the saline-exposed rats, the E17 MAM-exposed rats exhibited spontaneous hyperactivity that emerged only after puberty. At adulthood, they also exhibited hypersensitivity to the locomotor activating effects of a mild stress and a glutamatergic N-methyl-D-aspartate receptor antagonist (MK-801), as well as PPI deficits whereas before puberty no perturbations were observed. In addition, spatial working memory did not undergo the normal peri-pubertal maturation seen in the sham rats. Social interaction deficits were observed in MAM rats, at both pre- and post-puberty. Our study further confirms that transient prenatal disruption of neurogenesis by MAM at E17 is a valid behavioral model for schizophrenia as it is able to reproduce some fundamental features of schizophrenia with respect to both phenomenology and temporal pattern of the onset of symptoms and deficits.