The binding sites for benztropines and dopamine in the dopamine transporter overlap

Analogs of benztropines (BZTs) are potent inhibitors of the dopamine transporter (DAT) but are less effective than cocaine as behavioral stimulants. As a result, there have been efforts to evaluate these compounds as leads for potential medication for cocaine addiction. Here we use computational modeling together with site-directed mutagenesis to characterize the binding site for BZTs in DAT. Docking into molecular models based on the structure of the bacterial homolog LeuT supported a BZT binding site that overlaps with the substrate-binding pocket. In agreement, mutations of residues within the pocket, including² Val152^3.46 to Ala or Ile, Ser422^8.60 to Ala and Asn157^3.51 to Cys or Ala, resulted in decreased affinity for BZT and the analog JHW007, as assessed in [³H]dopamine uptake inhibition assays and/or [³H]CFT competition binding assay. A putative polar interaction of one of the phenyl ring fluorine substituents in JHW007 with Asn157^3.51 was used as a criterion for determining likely binding poses and establish a structural context for the mutagenesis findings. The analysis positioned the other fluorine-substituted phenyl ring of JHW007 in close proximity to Ala479^10.51/Ala480^10.52 in transmembrane segment (TM) 10. The lack of such an interaction for BZT led to a more tilted orientation, as compared to JHW007, bringing one of the phenyl rings even closer to Ala479^10.51/Ala480^10.52. Mutation of Ala479^10.51 and Ala480^10.52 to valines supported these predictions with a larger decrease in the affinity for BZT than for JHW007. Summarized, our data suggest that BZTs display a classical competitive binding mode with binding sites overlapping those of cocaine and dopamine.     

耳蜗血管纹钾钠氯化合物协同转运子对年龄相关性听力下降的影响

目的 观察具有年龄相关性听力下降（age-related hearing loss，AHL）特点的不同周龄C57BL／6J鼠和钾钠氯化合物协同联合转运子-1（Na-K-2Cl cotransporter-1，NKCC1）基因敲除鼠年龄相关性听力改变与耳蜗血管纹NKCC1表达的情况，并检测不同龄的NKCC1^＋/-杂合子小鼠耳蜗NKCC1蛋白表达及耳蜗扫描电镜形态变化，研究血管纹NKCC1在老年性聋发病中的作用。方法 应用听性脑于反应（ABR）分别检测4、8、16、32、48、60周龄组C57BL／6J小鼠和两种基因型NKCC1小鼠的听力，采用免疫组化法观察其血管纹NKCC1表达的变化，同时采用免疫印迹杂交（Western Blot）检测不同周龄小鼠耳蜗的NKCC1蛋白的含量，扫描电镜观察不同周龄小鼠的耳蜗基底膜结构。结果 C57BL／6J小鼠随鼠龄增加而出现听力下降，自16周龄时ABR阈值出现显著性增高（P〈0．05）；血管纹NKCC1蛋白表达也呈现随鼠龄增加而逐步减低，其灰度值自16周龄时显著增高（P〈0．01）。NKCC1^＋/-杂合子鼠的听力随鼠龄增长而逐渐下降，老龄小鼠的ABR阈值与其低龄时相比，阈值显著性升高（P〈0．05）。老龄小鼠的耳蜗NKCC1蛋白含量低于低龄小鼠，老龄NKCC1^＋／-小鼠的耳蜗底回外毛细胞（OHC）出现散在性点状缺失。结论 AHL小鼠血管纹NKCC1蛋白表达随年龄增长而减少，显示与AHL相关，老年性聋的发病可能与血管纹NKCC1通道蛋白的遗传特性及功能有关。     

Empagliflozin治疗2型糖尿病达到HbA1c＜7.0％的有效性及安全性评价

目的 系统评价钠葡萄糖协同转运蛋白2 (SGLT2)抑制剂empagliflozin治疗2型糖尿病(T2DM)达到HbA1c＜7.0％的有效性与安全性.方法 计算机检索Pubmed、Cochrane Library、EMbase,CBM和CNKI数据库发表empagliflozin的文献,检索时限均为建库至2015年10月,筛选合格随机对照试验(RCT).根据Cochrane系统评价手册5.1评估纳入研究偏倚风险,采用RevMan 5.3软件进行统计分析.结果 纳入8篇RCT,共4 728例研究对象.结果显示:empagliflozin使HbA1c＜7.0％的达标率高于安慰剂(10 mg RR=2.68,95％CI:2.06～3.47,P＜0.05;25 mg RR=3.25,95％CI:2.49～4.25,P＜0.05),且不增加低血糖风险(10 mg RR=1.04,95％CI:0.89～1.21,P＞0.05;25 mg RR=1.06,95％CI:0.91～1.23,P＞0.05),同时能降低体质量(10 mg WMD=-1.75,95％CI:—1.96～—1.55,P＜0.05;25 mg WMD=—1.95,95％CI:—2.16～—1.74,P＜0.05),收缩压和舒张压及改善空腹血糖;但增加了生殖道感染风险,在发生泌尿道感染和严重不良事件方面差异无统计学意义,且对T2DM患者肾功能无影响及不增加全因死亡率.结论 Empagliflozin能够有效地使T2DM患者达到HbA1c＜7.0％,另有减轻体质量和降压的临床获益,且安全性好.     

氯离子转运通道在人视网膜色素上皮细胞吞噬过程中的作用

目的：探讨丁脲胺或DNDS作用下视网膜色素上皮（RPE）细胞顶端膜和基底膜上氯离子转运通道对RPE吞噬功能的影响,阐明氯离子转运通道参与PRE吞噬作用的机制。方法:利用微孔滤膜培养系统体外培养极性化单层RPE细胞,以荧光包被的乳胶株作为吞噬标记物,建立RPE细胞吞噬模型,分别以1、5、10、50、100、500和1 000 μmol·L^-1丁脲胺,以及0.01、.10、0.50、1.00、3.00、5.00和10.00 mmol·L^-1DNDS作用RPE细胞6 h后,应用流式细胞仪定量检测RPE细胞吞噬指数。结果:不同浓度丁脲胺作用于RPE细胞顶端膜或基底膜,RPE细胞对乳胶株的吞噬指数与对照组比较差异无显著性（P>0.05）;0.01和0.10μmol·L-1DNDS作用于RPE细胞基底膜,RPE细胞对乳胶株的吞噬指数与对照组比较差异无显著性（P>0.05）,而0.50、1.00、3.00、5.00、10.00 mmol·L^-1的DNDS作用于RPE细胞基底膜,RPE细胞对乳胶株的吞噬指数降低（P<0.05或P<0.01）,并随着浓度的增加,吞噬指数逐渐降低;但同样浓度的DNDS作用于RPE细胞顶端膜,RPE细胞对乳胶株的吞噬指数无明显改变（P>0.05）。结论:顶端膜的丁脲胺敏感性Na⁺-K⁺-2Cl^－协同转运载体不参与RPE吞噬乳胶株的过程,而基底膜的DNDS敏感性Cl－/HCO₃^－交换蛋白及氯离子通道在DNDS作用后,对人RPE细胞非特异性吞噬过程发挥抑制作用。     

Changes in urinary excretion of water and sodium transporters during amiloride and bendroflumethiazide treatment

AIM:To quantify changes in urinary excretion of aquaporin2 water channels(u-AQP2),the sodiumpotassium-chloride co-transporter(u-NKCC2) and the epithelial sodium channels(u-ENa C) during treatment with bendroflumethiazide(BFTZ),amiloride and placebo.METHODS:In a randomized,double-blinded,placebocontrolled,3-way crossover study we examined 23 healthy subjects on a standardized diet and fluid intake.The subjects were treated with amiloride 5 mg,BFTZ 1.25 mg or placebo twice a day for 4.5 d before each examination day.On the examination day,glomerular filtration rate was measured by the constant infusion clearance technique with 51Cr-EDTA as reference substance.To estimate the changes in water transport via AQP2 and sodium transport via NKCC2 and ENa C,u-NKCC2,the gamma fraction of ENa C(u-ENa Cγ),andu-AQP2 were measured at baseline and after infusion with 3% hypertonic saline.U-NKCC2,u-ENa Cγ,u-AQP2 and plasma concentrations of vasopressin(p-AVP),renin(PRC),angiotensin Ⅱ(p-ANG Ⅱ) and aldosterone(p-Aldo) were measured,by radioimmunoassay.Central blood pressure was estimated by applanation tonometry and body fluid volumes were estimated by bio-impedance spectroscopy.General linear model with repeated measures or related samples Friedman's two-way analysis was used to compare differences.Post hoc Bonferroni correction was used for multiple comparisons of post infusion periods to baseline within each treatment group.RESULTS:At baseline there were no differences in u-NKCC2,u-ENa Cγ and u-AQP2.PRC,p-Ang Ⅱ and p-Aldo were increased during active treatments(P 0.001).After hypertonic saline,u-NKCC2 increased during amiloride(6% ± 34%;P = 0.081) and increased significantly during placebo(17% ± 24%;P = 0.010).U-AQP2 increased significantly during amiloride(31% ± 22%;P 0.001) and placebo(34% ± 27%;P 0.001),while u-NKCC2 and u-AQP2 did not change significantly during BFTZ(-7% ± 28%;P = 0.257 and 5% ± 16%;P = 0.261).U-ENa Cγ increased in all three groups(P 0.050).PRC,AngⅡ and p-Aldo decreased to the same extent,while AVP increased,but to a smaller degree during BFTZ(P = 0.048).c DBP decreased significantly during BFTZ(P 0.001),but not during amiloride or placebo.There were no significant differences in body fluid volumes.CONCLUSION:After hypertonic saline,u-NKCC2 and u-AQP2 increased during amiloride,but not during BFTZ.Lower p-AVP during BFTZ potentially caused less stimulation of NKCC2 and AQP2 and subsequent lower reabsorption of water and sodium.     

钾-氯协同转运子1新的同分异构体的发现及其转录调控意义

目的 寻找钾-氯协同转运子(KCC)1新的同分异构体及分析其意义。方法 抽提正常人肾组织RNA，用已发表KCC1序列设计引物，进行cDNA末端陕速放大(RACE)及RT-PCR，以Northern印迹分析证实新的同分异构体的存在，并用生物信息学方法比较新序列。结果 RACE和Northern印迹分析结果证实在人肾组织中有3种KCC1的新的同分异构体，与野生型KCC1不同的是，新的KCC1同分异构体2含部分内含子1的序列(外显子1b)，作为其外显子1，蛋白翻译起始密码子也位于该外显子中。新的KCC1同分异构体3含外显子1b、外显子2、内含子2及以后与野生型KCC1相同的序列，蛋白翻译起始密码子位于外显子4，导致一长的5’端非翻译区，内含子2靠外显子3的位置有一翻译终止密码子。新的KCC1同分异构体4含部分内含子3作为其5’端非翻译区。结论 新的异构体的发现为KCC家族增添了新的成员，并为KCC1这一重要分子的转录和功能调节提供了新的线索，其特异性序列可用于分析它们在人类疾病中的病理意义。由于启动子类型的不同对同分异构体基因转录的选择起主动的调节作用．多个同分异构体及多个启动子的存在对理解KCC1复杂的生理功能、调节过程和致病机制具有重要意义。     

Mechanism for alternating access in neurotransmitter transporters

Crystal structures of LeuT, a bacterial homologue of mammalian neurotransmitter transporters, show a molecule of bound substrate that is essentially exposed to the extracellular space but occluded from the cytoplasm. Thus, there must exist an alternate conformation for LeuT in which the substrate is accessible to the cytoplasm and a corresponding mechanism that switches accessibility from one side of the membrane to the other. Here, we identify the cytoplasmic accessibility pathway of the alternate conformation in a mammalian serotonin transporter (SERT) (a member of the same transporter family as LeuT). We also propose a model for the cytoplasmic-facing state that exploits the internal pseudosymmetry observed in the crystal structure. LeuT contains two structurally similar repeats (TMs1–5 and TMs 6–10) that are inverted with respect to the plane of the membrane. The conformational differences between them result in the formation of the extracellular pathway. Our model for the cytoplasm-facing state exchanges the conformations of the two repeats and thus exposes the substrate and ion-binding sites to the cytoplasm. The conformational change that connects the two states primarily involves the tilting of a 4-helix bundle composed of transmembrane helices 1, 2, 6, and 7. Switching the tilt angle of this bundle is essentially equivalent to switching the conformation of the two repeats. Extensive mutagenesis of SERT and accessibility measurements, using cysteine reagents, are accommodated by our model. These observations may be of relevance to other transporter families, many of which contain internal inverted repeats.     

二甲双胍对甲状腺功能减退大鼠甲状腺激素的影响

目的 探讨二甲双胍对甲状腺功能减退大鼠甲状腺激素水平的影响及其机制.方法 取3个月龄雄性SD大鼠36只,随机分为正常对照(NC)组、甲状腺功能减退(HT)组、二甲双胍(Met)组.HT组和Met组以含0.01％氨基三唑的饮用水喂养大鼠4周制备甲状腺功能减退大鼠模型,NC组大鼠喂养普通饮用水.造模成功后,Met组用200 mg/kg二甲双胍灌胃,HT组和NC组用等体积0.05％羟甲基纤维素钠(CMC-Na)灌胃.于灌胃第8周时对3组大鼠甲状腺组织行H-E染色,采用qPCR检测下丘脑促甲状腺激素释放激素前体(proTRH) mRNA的表达;于灌胃第8、12周时采用电化学发光法检测3组大鼠血清T3、T4水平,采用qPCR和蛋白质印迹法检测甲状腺组织钠碘同向转运体(NIS) mRNA和蛋白表达.结果 在灌胃8、12周时,HT组大鼠血清T3、T4水平均低于NC组,而Met组T3、T4水平均高于HT组(P＜0.05).灌胃8周时,H-E染色结果显示NC组滤泡大小不等,内见大量胶质及吸收空泡;HT组大鼠甲状腺滤泡缩小,滤泡上皮成柱状,滤泡周围吸收空泡减少;Met组滤泡上皮增生较HT组稍改善.灌胃8周时,qPCR结果显示HT组大鼠下丘脑proTRH和甲状腺组织NIS mRNA表达均高于NC组,而Met组均低于HT组(P＜0.05);蛋白质印迹法检测结果显示Met组NIS蛋白表达低于HT组(P＜0.05),而HT组和NC组NIS蛋白表达差异无统计学意义(P＞0.05).灌胃12周时,HT组和Met组甲状腺组织NIS mRNA表达差异无统计学意义(P＞0.05),Met组NIS蛋白表达高于HT组(P＜0.05).结论 二甲双胍能升高甲状腺功能减退大鼠的甲状腺激素水平,但短期内并不是由甲状腺NIS表达升高所致.     

人甲状腺原代细胞的长期培养

目的　探索人甲状腺细胞长期原代培养的方法及细胞在不同时期的功能变化。方法　常规分离甲状腺细胞,并用含有10%胎牛血清(FBS)、谷氨酰胺、牛胰岛素、10mU/LTSH和氢化可的松的MEM培养,细胞铺成单层后换用4% FBC的培养基培养,分别在不同时间观察甲状腺细胞吸碘率、细胞生长情况,免疫组化观察甲状腺特异抗原甲状腺球蛋白(Tg)表达水平,RT PCR测定钠碘转运子(NIS)基因表达水平。结果　甲状腺细胞在培养40d左右仍生长良好,培养14d细胞具有80%的吸碘率,培养7dTg表达可达95%,培养14dTg表达仍达60%,NISmRNA表达在培养7d达98%,而培养14d表达率只有40%。结论　应用该研究所建立的培养条件可以使甲状腺细胞生存40d以上,该实验条件下培养3, 7, 14,28d的原代甲状腺滤泡上皮细胞以培养7d的细胞NISmRNA,Tg表达、摄碘功能最接近基线水平。     

A Convergent Functional Genomics Analysis to Identify Biological Regulators Mediating Effects of Creatine Supplementation

Creatine (Cr) and phosphocreatine (PCr) are physiologically essential molecules for life, given they serve as rapid and localized support of energy- and mechanical-dependent processes. This evolutionary advantage is based on the action of creatine kinase (CK) isozymes that connect places of ATP synthesis with sites of ATP consumption (the CK/PCr system). Supplementation with creatine monohydrate (CrM) can enhance this system, resulting in well-known ergogenic effects and potential health or therapeutic benefits. In spite of our vast knowledge about these molecules, no integrative analysis of molecular mechanisms under a systems biology approach has been performed to date; thus, we aimed to perform for the first time a convergent functional genomics analysis to identify biological regulators mediating the effects of Cr supplementation in health and disease. A total of 35 differentially expressed genes were analyzed. We identified top-ranked pathways and biological processes mediating the effects of Cr supplementation. The impact of CrM on miRNAs merits more research. We also cautiously suggest two dose–response functional pathways (kinase- and ubiquitin-driven) for the regulation of the Cr uptake. Our functional enrichment analysis, the knowledge-based pathway reconstruction, and the identification of hub nodes provide meaningful information for future studies. This work contributes to a better understanding of the well-reported benefits of Cr in sports and its potential in health and disease conditions, although further clinical research is needed to validate the proposed mechanisms.