利拉鲁肽治疗2型糖尿病的临床研究进展

利拉鲁肽是一种新型胰高糖素样肽-1 GLP-1类似物,能迅速、高效、持久地降低血糖及糖化血红蛋白,具有改善胰岛β细胞功能、调节收缩压、保护心血管、降低血脂、减轻体重、延迟胃排空、增加饱腹感等作用。此外,利拉鲁肽具有葡萄糖依赖的促胰岛素分泌作用,仅在机体血糖水平高时刺激胰岛素的释放,但当患者体内血糖浓度恢复至正常时,GLP-1分泌促胰岛素的作用便消退,因而发生低血糖事件的几率较低,能有效地改善糖尿病患者的病情,已成为目前2型糖尿病治疗领域的研究热点。本文主要就利拉鲁肽治疗2型糖尿病的临床研究进展进行综述。     

2型糖尿病患者早期使用胰高血糖素样受体激动剂

糖尿病目前已成为继心血管疾病和肿瘤之后的第三位主要非传染性疾病,其中90%为2型糖尿病患者。胰高血糖素样肽-1类似物(GLP-I类似物)作为一种新型的降糖药物,具有降低体重、降低收缩压、改善胰岛细胞功能,已成为2型糖尿病治疗的新热点。艾塞那肽和利拉鲁肽作为肠促胰素激素,与人体内天然GLP-1保持了高度同源性(97%)。近几年来受到人们广泛关注。本综述针对2型糖尿病患者早期使用胰岛素样受体激动剂艾塞那肽和利拉鲁肽的安全性和有效性进行评估。     

酪酸梭菌活菌胶囊联合利拉鲁肽对2型糖尿病患者的疗效及对肠道菌群的影响

目的探究酪酸梭菌活菌胶囊联合利拉鲁肽对2型糖尿病患者的治疗效果及其对肠道菌群的影响,为该类患者的治疗提供参考。方法选取2017年11月至2019年3月于我院接受治疗的120例2型糖尿病患者为研究对象,随机分为利拉鲁肽组和联合治疗组,各60例。利拉鲁肽组患者使用利拉鲁肽进行治疗,联合治疗组患者使用酪酸梭菌活菌胶囊联合利拉鲁肽进行治疗。采用血糖检测仪对两组患者空腹血糖(FBG)、餐后两小时血糖(2hPG)、糖化血红蛋白(HbA1c)水平进行检测。采用酶联免疫吸附实验检测总胆固醇(TC)、三酰甘油(TG)、血清趋化素(Chemerin)水平。采用免疫透射比浊法检测谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)、丙二醛(MDA)水平,使用光冈法对两组患者肠道菌群进行检测,对比两组患者治疗效果。结果治疗半年后联合治疗组患者FBG、2hPG、HbA1c水平均低于利拉鲁肽组(均P0.05)。治疗后利拉鲁肽组患者TC、TG、Chemerin水平均低于联合治疗组(均P0.05)。治疗后联合治疗组患者血清GSH-Px、SOD水平高于利拉鲁肽组,MDA水平低于利拉鲁肽组(均P0.05)。治疗后联合治疗组患者肠道乳杆菌、双歧杆菌数量高于利拉鲁肽组,肠球菌、肠杆菌数量低于利拉鲁肽组(均P0.05)。联合治疗组患者治疗总有效率显著高于利拉鲁肽组(95.00%vs 83.33%,χ~2=4.227,P=0.040)。结论酪酸梭菌活菌胶囊联合利拉鲁肽能够改善2型糖尿病患者糖代谢指标水平以及血脂水平,提升患者抗氧化能力,调节患者肠道菌群,治疗效果显著。     

利拉鲁肽联合维生素D对高脂诱导非酒精性脂肪肝小鼠的影响及机制

目的: 探讨利拉鲁肽联合维生素D对高脂诱导非酒精性脂肪肝(NAFLD)小鼠的影响及其潜在机制。方法: C57BL/6小鼠随机平均分为对照组、NAFLD模型组、利拉鲁肽组、维生素D组和利拉鲁肽联合维生素D组,每组10只。对照组普通饲料连续喂养12 周; 模型组高脂饲料连续喂养12周; 利拉鲁肽组、维生素D组和联合组均高脂饲料连续喂养12周, 从第9周起上述3组小鼠分别腹腔注射0.6 mg/(kg·d)利拉鲁肽、灌胃250 mg/(kg·d)维生素D和腹腔注射0.6 mg/(kg·d)利拉鲁肽及灌胃250 mg/(kg·d)维生素D。喂养至12周后,收集各组小鼠血液和肝组织进行生化及病理检测;并采用免疫印迹方法检测各组小鼠肝组织AMP活化蛋白激酶(AMPK)磷酸化水平。结果: 与模型组相比,利拉鲁肽或维生素D单独或联合治疗均可改善NAFLD小鼠肝脏脂质积累(甘油三酯: 6.0±0.7 vs 3.8±0.3, 3.9±0.3和2.1±0.2,P均＜0.05;胆固醇:1.4±0.5 vs 0.9±0.2, 0.8±0.2和0.5±0.1,P均＜0.05)和脂肪变性(NAFLD活动评分:2.4±0.3 vs 1.0±0.2, 0.9±0.1和0.6±0.1,P均＜0.05);此外与利拉鲁肽或维生素D组相比,利拉鲁肽联合维生素D治疗效果更明显,而且可能与调节胰岛素抵抗和AMPK磷酸化相关。结论: 结果表明,维生素D可增强利拉鲁肽对高脂诱导NAFLD的治疗效果,其机制可能与调节胰岛素抵抗和AMPK磷酸化有关。     

利拉鲁肽抑制高糖诱导的小鼠胰岛细胞损伤

利拉鲁肽(liraglutide, Lira)是胰高血糖素样肽-1的类似物,在糖尿病治疗中发挥重要作用,但利拉鲁肽通过改善胰岛β细胞的功能实现治疗糖尿病的具体机制尚未完全阐明。本研究采用高糖(33 mmol/L)诱导胰岛MIN6细胞48 h建立高糖损伤模型,CCK-8检测发现,与对照组相比,高糖组MIN6细胞活力下降(P<0.05),利拉鲁肽作用高糖组细胞活力升高(P<0.05);小鼠胰岛素和ATP含量检测发现,与对照组相比,高糖组胰岛素分泌降低(P<0.01),ATP含量减少(P<0.001),利拉鲁肽作用高糖组胰岛素释放量增加(P<0.05)和细胞内ATP含量增加(P<0.001);采用活体细胞线粒体膜通道孔(MPTP)荧光检测发现,与对照组相比,高糖组绿色荧光强度降低(P<0.001),利拉鲁肽作用高糖组绿色荧光强度增加(P<0.001);DCFH-DA探针联合流式细胞仪检测细胞活性氧簇(ROS)含量发现,与对照组相比,高糖组ROS水平升高(P<0.001),利拉鲁肽作用高糖组ROS水平降低(P<0.01);细胞内丙二醛...     

贝那鲁肽与利拉鲁肽对2型糖尿病患者长期结局影响的对比研究

摘要 目的：对比分析贝那鲁肽与利拉鲁肽对2型糖尿病患者长期结局的影响，为二甲双胍和磺脲类药物治疗后血糖不能达标的患者应用胰高糖素样肽-1（GLP-1） 受体激动剂提供临床依据。方法：选取2018年3月～2019年3月在我院就诊的使用二甲双胍或者二甲双胍联合磺脲类药物血糖未达标的2型糖尿病患者150例作为研究对象，随机分为观察组和对照组，每组各75例。观察组使用贝那鲁肽注射液治疗，对照组使用利拉鲁肽注射液治疗，所有患者均连续治疗24周。比较两组患者治疗前后空腹血糖（FPG）、餐后2 h血糖（2hPG）、糖化血红蛋白（HbA1c）、收缩期血管峰值血流( PSV)、血管内膜中层厚度(IMT) 、肌酐（Scr）和尿素氮（BUN）水平，两年内的不良事件发生率、心血管相关死亡率和全因死亡率。结果：两组患者之间治疗前、后FPG、2hPG、HbA1c、PSV、IMT、Scr和BUN水平无统计学差异（P>0.05）, 观察组的不良事件总发生率（8.00%）较对照组（20.00%）明显降低（P<0.05），两组患者心血管相关死亡率和全因死亡率相比，差异无统计学意义（P>0.05）。结论：在使用二甲双胍或者二甲双胍联合磺脲类药物血糖未达标的2型糖尿病患者中，与利拉鲁肽相比，贝那鲁肽在降低血糖、改善下肢血管功能、肾功能和死亡率方面无明显差异，但不良事件发生率更低。     

茵陈五苓散联合利拉鲁肽对肥胖型2型糖尿病患者糖脂代谢、胰岛素敏感性和氧化应激的影响

摘要 目的：探讨茵陈五苓散联合利拉鲁肽对肥胖型2型糖尿病（T2DM）患者糖脂代谢、胰岛素敏感性和氧化应激的影响。方法：选取2021年12月~2022年12月期间广州中医药大学附属佛山中医院收治的160例痰湿内蕴型肥胖T2DM患者。根据随机数字表法将患者分为对照组（利拉鲁肽治疗,80例）和研究组（茵陈五苓散联合利拉鲁肽治疗,80例）。对比两组疗效、糖脂代谢指标、肥胖相关指标、胰岛素敏感性、氧化应激和不良反应发生情况。结果：与对照组相比,研究组的临床总有效率更高（P<0.05）。研究组治疗后体质量指数（BMI）、空腹血糖（FBG）、腰臀比（WHR）、餐后2 h血糖（2hPG）、丙二醛（MDA）、糖化血红蛋白（HbAlc）、稳态模型胰岛素抵抗指数（HOMA-IR）、总胆固醇（TC）、黄嘌呤氧化酶（XO）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）较对照组低（P<0.05）。治疗后研究组稳态模型胰岛β细胞功能指数（HOMA-β）、高密度脂蛋白胆固醇（HDL-C）、超氧化物歧化酶（SOD）高于对照组（P<0.05）。两组不良反应发生率组间对比未见差异（P>0.05）。结论：茵陈五苓散联合利拉鲁肽治疗肥胖型T2DM患者疗效确切,可调节糖脂代谢水平,降低胰岛素敏感性,减轻氧化应激,且具有一定安全性,值得临床借鉴应用。     

利拉鲁肽联合二甲双胍对肥胖合并2型糖尿病患者糖脂代谢、胰岛β细胞功能及体脂的影响探究

目的探讨利拉鲁肽联合二甲双胍对肥胖合并2型糖尿病患者糖脂代谢、胰岛β细胞功能及体脂的影响。 方法选择成都市郫都区第二人民医院2017年1月至2018年12月收治的肥胖合并2型糖尿病患者141例作为研究对象,按照随机数字表法分为利拉鲁肽组47例、二甲双胍组47例与联合组47例。利拉鲁肽组采用利拉鲁肽治疗,二甲双胍组采用二甲双胍治疗,联合组采用利拉鲁肽联合二甲双胍治疗。三组疗程均为3个月。比较三组治疗前后糖代谢、脂代谢、胰岛β细胞功能和体重指数（BMI）变化,及不良反应,治疗前后比较采用配对t检验,三组间比较采用F检验,两组间比较采用LSD-t检验。 结果三组治疗后FPG、HbA1c和2hPG水平较治疗前降低,差异具有统计学意义（P均< 0.05）;联合组治疗后FPG（6.57±0.39）?mmol/L、HbA1c（7.03±0.42）%和2hPG（8.78±0.45）mmol/L低于利拉鲁肽组（7.03±0.32）mmol/ L、（7.68±0.35）%、（9.56±0.65）mmol/L（t = 6.251、8.151、6.764,P均?< 0.05）和二甲双胍组（7.06±0.39）mmol/L、（7.76±0.46）%、（9.70±0.81）?mmol/L,差异具有统计学意义（t = 6.091、8.034、6.807,P均< 0.05）。三组治疗后HDL-C水平较治疗前升高,而TC、LDL-C和TG水平较治疗前降低,差异具有统计学意义（P均< 0.05）;联合组治疗后HDL-C （1.56±0.13） mmol/ L高于利拉鲁肽组（1.29±0.14） mmol/ L（t = 9.689,P < 0.05）和二甲双胍组（1.32±0.15） mmol/ L,差异具有统计学意义（t = 8.289,P < 0.05）;而联合组TC（4.35±0.38） mmol/L、LDL-C（2.79±0.21）mmol/L和TG（2.15±0.26） mmol/ L低于利拉鲁肽组（5.18±0.43）mmol/L、（3.19±0.15）mmol/L、（2.65±0.17） mmol/L（t = 9.916、10.626、11.035,P < 0.05）和二甲双胍组（5.15±0.34）mmol/L、（3.23±0.25）mmol/ L、（2.68±0.23） mmol/ L,差异具有统计学意义（t = 10.756、9.239、10.467,P均< 0.05）。三组治疗后HOMA-β较治疗前升高,差异具有统计学意义（P < 0.05）;联合组治疗后HOMA-β（4.87±0.28）高于利拉鲁肽组（4.15±0.36）和二甲双胍组（4.08±0.41）,差异具有统计学意义（t = 10.823,10.909,P均< 0.05）。三组治疗后BMI较治疗前降低,差异具有统计学意义（P < 0.05）;联合组治疗后BMI （28.08±0.37）kg/m²低于利拉鲁肽组（29.73±0.49）kg/m²和二甲双胍组（29.61±0.43）kg/m²,差异具有统计学意义（t = 18.423,18.490,P均< 0.05）。 结论利拉鲁肽联合二甲双胍对肥胖合并2型糖尿病患者效果良好,可改善患者糖脂代谢和胰岛β细胞功能,降低体质指数,值得临床借鉴。     

加味黄连解毒汤联合利拉鲁肽对初发2型糖尿病肥胖患者糖脂代谢、炎症反应及胰岛功能的影响

摘要 目的：探讨加味黄连解毒汤联合利拉鲁肽对初发2型糖尿病肥胖患者糖脂代谢、炎症反应及胰岛功能的影响。方法：选取2016年1月至2018年5月复旦大学附属华山医院北院收治的104例初发2型糖尿病肥胖患者,按照随机数字表法将患者分为对照组（n=52）和研究组（n=52）。对照组给予利拉鲁肽治疗,研究组给予加味黄连解毒汤联合利拉鲁肽治疗,两组均治疗3个月。比较两组治疗前、治疗3个月后体质量指数（BMI）、血糖指标[空腹血糖（FPG）、餐后2小时血糖（2hPG）、糖化血红蛋白（HbA1c）]、血脂指标[总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）]、炎症指标[C反应蛋白（CRP）、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）]及胰岛功能指标[胰岛素抵抗指数（HOMA-IR）、胰岛β细胞功能指数（HOMA-β）],并记录两组治疗过程中不良反应发生情况。结果：治疗3个月后,两组的BMI、FPG、2hPG、HbA1c、HOMA-IR及血清TG、TC、LDL-C、CRP、TNF-α、IL-6水平均低于治疗前,HOMA-β及血清HDL-C水平均高于治疗前,且研究组BMI、FPG、2hPG、HbA1c、HOMA-IR及血清TG、TC、LDL-C、CRP、IL-6、TNF-α水平均较对照组降低,血清HDL-C水平及HOMA-β较对照组升高（P<0.05）。两组不良反应发生率比较无差异（P>0.05）。结论：加味黄连解毒汤联合利拉鲁肽治疗初发2型糖尿病肥胖患者安全有效,能够减轻患者体重,改善血糖、血脂水平以及胰岛功能,减轻炎症反应。     

利拉鲁肽联合达格列净对超重或肥胖2型糖尿病患者肾功能、氧化应激以及内脏脂肪含量的影响

摘要 目的：探讨利拉鲁肽联合达格列净对超重或肥胖2型糖尿病（T2DM）患者肾功能、氧化应激以及内脏脂肪含量的影响。方法：选取我院于2018年1月~2020年5月期间接收的108例超重或肥胖T2DM患者,按照随机数字表法分为对照组（n=54）和观察组（n=54）。对照组给予利拉鲁肽治疗,观察组给予利拉鲁肽联合达格列净治疗,均治疗12周。对比两组肾功能[血胱抑素 C（CysC）、血肌酐（Scr）、血尿酸（SUA）]、氧化应激[丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-PX）、超氧化物歧化酶（SOD）]、体质量指数（BMI）、腰围、血糖指标[空腹血糖（FBG）、餐后2 h血糖（2hPBG）、糖化血红蛋白（HbA1c）]以及体成分指标（全身脂肪百分比、内脏脂肪含量）,记录两组治疗期间不良反应情况。结果：两组治疗后BMI、腰围、2hPBG、FBG、HbA1c均下降,且观察组较对照组低（P<0.05）。两组治疗后MDA均下降,且观察组较对照组低（P<0.05）,两组治疗后SOD、GSH-PX均升高,且观察组较对照组高（P<0.05）。两组治疗后全身脂肪百分比、内脏脂肪含量均下降,且观察组较对照组低（P<0.05）。两组治疗前后CysC、Scr、SUA组内及组间对比均无统计学差异（P>0.05）。两组不良反应发生率对比无统计学差异（P>0.05）。结论：超重或肥胖T2DM患者利拉鲁肽治疗基础上联合达格列净,降糖效果确切,减轻机体氧化应激,降低内脏脂肪含量,对肾功能无显著影响,且不增加不良反应发生率。     

Flow cytometric analysis reveals the presence of asialo GM1 on the surface membrane of alloimmune cytotoxic T lymphocytes

Previous studies have demonstrated that natural killer (NK) cells express the glycolipid asialo GM1, as evidenced by the sensitivity of NK cells to treatment with anti-asialo GM1 serum and complement. Because alloimmune cytotoxic T lymphocytes (CTL) were found to be insensitive to treatment with anti-asialo GM1 serum and complement, it was concluded that asialo GM1 is expressed by NK but not by CTL. However, fluorescence studies indicated that a significant proportion of peripheral T cells did express asialo GM1. Flow cytometric studies were undertaken to determine the extent to which alloimmune CTL express asialo GM1. Affinity-purified, monospecific IgG anti-asialo GM1 antibodies were used to label cells from mixed lymphocyte cultures. Separation of asialo GM1-positive and -negative fractions by cell sorting revealed that the majority of CTL activity resides in the asialo GM1-positive population. When these studies are compared with similar studies of splenic NK activity, it is apparent that, despite the relative insensitivity of CTL to treatment with anti-asialo GM1 and complement, both CTL and NK activity are enriched in the asialo GM1-positive cell population obtained by cell sorting.     

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.     

Association of common polymorphisms in the IL2RA gene with type 1 diabetes: evidence of 32,646 individuals from 10 independent studies

Single nucleotide polymorphisms (SNPs) in the interleukin 2 receptor alpha (IL2RA) gene have been suggested to be associated with type 1 diabetes (T1D) susceptibility. However, the results from individual studies are inconsistent. To explore the association of IL2RA polymorphisms with T1D, including rs11594656, rs2104286, rs3118470, rs41295061 and rs706778, a meta‐analysis involving 10 independent studies with 19 outcomes was conducted: five studies with a total of 10,572 cases and 12,956 controls were analysed for rs11594656 with T1D risk, three studies with 7300 cases and 8331 controls for rs2104286, three studies with 3880 cases and 5409 controls for rs3118470, five studies with 11,253 cases and 13,834 controls for rs41295061 and three studies with 1896 cases and 1709 controls for rs706778 respectively. Using minor allelic comparison, the five investigated SNPs were all observed to have a significant association with T1D: For rs11594656, fixed effect model (FEM) odds ratio (OR) 0.87, 95% confidence interval (CI) 0.83, 0.91; rs2104286, FEM OR 0.81, 95% CI 0.77, 0.85; rs3118470, FEM OR 1.23, 95% CI 1.16, 1.31; rs41295061, random effect model (REM) OR 0.67, 95% CI 0.60, 0.76 and rs706778 FEM OR 1.20, 95% CI 1.08, 1.33. Similar results were obtained when all the included studies were calculated by a REM. Our meta‐analysis suggests that all five SNPs in the IL2RA gene are risk factors for T1D risk, and rs11594656, rs2104286 and rs41295061 are the most associated SNPs in the populations investigated. This conclusion warrants confirmation by further studies.     

The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.     

Localization of caveolin-1 and c-src in mature and differentiating photoreceptors: raft proteins co-distribute with rhodopsin during development

Numerous biochemical and morphological studies have provided insight into the distribution pattern of caveolin-1 and the presence of membrane rafts in the vertebrate retina. To date however, studies have not addressed the localization profile of raft specific proteins during development. Therefore the purpose of our studies was to follow the localization pattern of caveolin-1, phospho-caveolin-1 and c-src in the developing retina and compare it to that observed in adults. Specific antibodies were used to visualize the distribution of caveolin-1, c-src, a kinase phosphorylating caveolin-1, and phospho-caveolin-1. The labeling pattern of this scaffolded complex was compared to those of rhodopsin and rhodopsin kinase. Samples were analyzed at various time points during postnatal development and compared to adult retinas. The immunocytochemical studies were complemented with immunoblots and immunoprecipitation studies. In the mature retina caveolin-1 and c-src localized mainly to the cell body and IS of photoreceptors, with only very weakly labeled OS. In contrast, phospho-caveolin-1 was only detectable in the OS of photoreceptors. During development we followed the expression and distribution profile of these proteins in a temporal sequence with special attention to the period when OS formation is most robust. Double labeling immunocytochemistry and immunoprecipitation showed rhodopsin to colocalize and co-immunoprecipitate with caveolin-1 and c-src. Individual punctate structures between the outer limiting membrane and the outer plexiform layer were seen at P10 to be labeled by both rhodopsin and caveolin-1 as well as by rhodopsin and c-src, respectively. These studies suggest that membrane raft specific proteins are co-distributed during development, thereby pointing to a role for such complexes in OS formation. In addition, the presence of small punctate structures containing caveolin-1, c-src and rhodopsin raise the possibility that these proteins may transport together to OS during development and that caveolin-1 exists predominantly in a phosphorylated form in the OS.     

Analysis of high-resolution HapMap of DTNBP1 (Dysbindin) suggests no consistency between reported common variant associations and schizophrenia

DTNBP1 was first identified as a putative schizophrenia-susceptibility gene in Irish pedigrees, with a report of association to common genetic variation. Several replication studies have reported confirmation of an association to DTNBP1 in independent European samples; however, reported risk alleles and haplotypes appear to differ between studies, and comparison among studies has been confounded because different marker sets were employed by each group. To facilitate evaluation of existing evidence of association and further work, we supplemented the extensive genotype data, available through the International HapMap Project (HapMap), about DTNBP1 by specifically typing all associated single-nucleotide polymorphisms reported in each of the studies of the Centre d'Etude du Polymorphisme Humain (CEPH)-derived HapMap sample (CEU). Using this high-density reference map, we compared the putative disease-associated haplotype from each study and found that the association studies are inconsistent with regard to the identity of the disease-associated haplotype at DTNBP1. Specifically, all five "replication" studies define a positively associated haplotype that is different from the association originally reported. We further demonstrate that, in all six studies, the European-derived populations studied have haplotype patterns and frequencies that are consistent with HapMap CEU samples (and each other). Thus, it is unlikely that population differences are creating the inconsistency of the association studies. Evidence of association is, at present, equivocal and unsatisfactory. The new dense map of the region may be valuable in more-comprehensive follow-up studies.     

Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

The human tRNA thiouridine modification protein (TUM1), also designated as 3-mercaptopyruvate sulfurtransferase (MPST), has been implicated in a wide range of physiological processes in the cell. The roles range from an involvement in thiolation of cytosolic tRNAs to the generation of H₂S as signaling molecule both in mitochondria and the cytosol. TUM1 is a member of the sulfurtransferase family and catalyzes the conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide. Here, we purified and characterized two novel TUM1 splice variants, designated as TUM1-Iso1 and TUM1-Iso2. The purified proteins showed similar kinetic behavior and comparable pH and temperature dependence. Cellular localization studies, however, showed a different localization pattern between the isoforms. TUM1-Iso1 is exclusively localized in the cytosol, whereas TUM1-Iso2 showed a dual localization both in the cytosol and mitochondria. Interaction studies were performed with the isoforms both in vitro using the purified proteins and in vivo by fluorescence analysis in human cells, using the split-EGFP system. The studies showed that TUM1 interacts with the l-cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, suggesting a dual function of TUM1 both in sulfur transfer for the biosynthesis of the molybdenum cofactor, and for the thiolation of tRNA. Our studies point to distinct roles of each TUM1 isoform in the sulfur transfer processes in the cell, with different compartmentalization of the two splice variants of TUM1.     

Mechanism of inactivation of plasminogen activator inhibitor-1 by a small molecule inhibitor

The inactivation of plasminogen activator inhibitor-1 (PAI-1) by the small molecule PAI-1 inhibitor PAI-039 (tiplaxtinin) has been investigated using enzymatic analysis, direct binding studies, site-directed mutagenesis, and molecular modeling studies. Previously PAI-039 has been shown to exhibit in vivo activity in various animal models, but the mechanism of inhibition is unknown. PAI-039 bound specifically to the active conformation of PAI-1 and exhibited reversible inactivation of PAI-1 in vitro. SDS-PAGE indicated that PAI-039 inactivated PAI-1 predominantly through induction of PAI-1 substrate behavior. Preincubation of PAI-1 with vitronectin, but not bovine serum albumin, blocked PAI-039 activity while analysis of the reciprocal experiment demonstrated that preincubation of PAI-1 with PAI-039 blocked the binding of PAI-1 to vitronectin. Together, these data suggest that the site of interaction of the drug on PAI-1 is inaccessible when PAI-1 is bound to vitronectin and may overlap with the PAI-1 vitronectin binding domain. This was confirmed by site-directed mutagenesis and molecular modeling studies, which suggest that the binding epitope for PAI-039 is localized adjacent to the previously identified interaction site for vitronectin. Thus, these studies provide a detailed characterization of the mechanism of inhibition of PAI-1 by PAI-039 against free, but not vitronectin-bound PAI-1, suggesting for the first time a novel pool of PAI-1 exists that is vulnerable to inhibition by inactivators that bind at the vitronectin binding site.     

Characterization of the beta-adrenergic receptor mediating secretion of parathyroid hormone

In vitro incubation studies with bovine parathyroid gland slices compared the relative responsiveness of parathyroid hormone (PTH) secretion to isoprotherenol, epinephrine or norepinephrine. Isoproterenol was the most potent and norepinephrine the least potent of the three stimuli, suggesting a beta 2 type of an adrenergic response. However in this in vitro system, tazalol, a selective beta 1 adrenergic agonist significantly stimulated PTH secretion, whereas terbutaline, a selective beta 2 agonist had no effect. In addition, practolol, a selective beta 1 adrenergic antagonist blocked isoproterenol- or tazolol-stimulated PTH secretion. In vivo studies in normal human subjects showed that injection of te nonselective beta agonist, isoproterenol, (0.15 mg s.c.) significantly increased, whereas injection of the selective beta 2 agonist, terbulatine (0.3 mg s.c.) had no effect on serum PTH levels. These latter studies with putative selective beta adrenergic agents suggest that the beta adrenergic receptor mediating PTH secretion is of the beta 1 type (in contrast to the studies above with nonselective agents). The studies suggest that the beta adrenergic receptor mediating PTH secretion apparently differs from the classical beta 1 receptor described in th myocardium or the classical beta 2 receptor described in the bronchial smooth muscle.