Scintillation proximity assay in lead discovery

Background: Scintillation proximity assay (SPA) is a homogeneous scintillant bead-based platform for the measurement of biological processes and plays an important role in the identification of active chemical entities in drug discovery. Objective: The design and development of solid-phase SPA approaches are examined and compared with alternative non-radiometric fluorescence-based technologies. Methods: This review provides background on the principle of SPA and its application to biomolecular interactions from a variety of biological sources. Conclusion: The SPA approach is well suited to the demands of commercial high volume automation and assay miniaturization for target-based high-throughput screening campaigns on synthetic and natural product libraries as well as for benchtop characterization and confirmation studies. In the near future, innovations in the way SPA and fluorescence-based screening strategies are multiplexed will improve our comprehensive understanding of cellular system biology and dramatically advance the lead discovery process for the treatment of complex target-related disorders.     

Induction of apoptosis by heat and γ-radiation in a human lymphoid cell line; role of mitochondrial changes and caspase activation

Purpose: The aim of the study was to investigate the molecular mechanisms involved in apoptosis of human promyelocytic cells (HL60) induced by hyperthermia and to compare this to radiation-induced apoptosis as a reference model.

Materials and methods: Apoptosis of HL60 cells was induced by heat-treatment (43°C during 1?h) or by γ-radiation (8?Gy) and followed at increasing time periods after treatment with Annexin V binding to phosphatidylserine (PS). The transition of the mitochondrial membrane potential (Δψ_m) was estimated by the extent of mitochondrial JC-1 uptake. Bcl-2 and Bax protein expression levels were monitored using fluorescent-labelled antibodies. Caspase activation was studied using a fluorochrome-labelled pan-caspase inhibitor (FLICA), which also allowed one to study the kinetics of the apoptotic cascade.

Results: After heat-treatment or irradiation of HL60 cells, a decreased Δψ_m as well as PS membrane expression were detectable after 8?h. Bcl-2 and Bax protein expression levels were decreased and increased, respectively, 1?h after heat-treatment or irradiation. The apoptotic rate of HL60 cells, as measured by the FLICA binding, was faster with heat-treatment as compared to γ-irradiation. Addition of a pan-caspase inhibitor prevented PS externalization after heat-treatment but not after irradiation. The presence of a pan-caspase inhibitor did not influence the decrease of Δψ_m both after heat-treatment and γ-irradiation. However, the addition of the specific caspase-2 inhibitor zVDVAD-fmk prevented the mitochondrial breakdown after heat-treatment. Inhibition of caspase-2 had no effect on the γ-irradiation induced apoptosis.

Conclusion: These results suggest that the commitment to apoptosis in HL60 cells after heat-treatment is started by mitochondrial membrane transition involving the Bcl-2 family members and is mainly executed in a caspase-dependent pathway. The results suggest that caspase-2 plays a key role in the heat-induced apoptosis.     

Inhibition of Notch signalling has ability to alter the proximal and distal TCR signalling events in human CD3+ αβ T-cells

The Notch signalling pathway is an important regulator of T cell function and is known to regulate the effector functions of T cells driven by T cell receptor (TCR). However, the mechanism integrating these pathways in human CD3⁺ αβ T cells is not well understood. The present study was carried out to investigate how Notch and TCR driven signalling are synchronized in human αβ T cells. Differential expression of Notch receptors, ligands, and target genes is observed on human αβ T cells which are upregulated on stimulation with α-CD3/CD28 mAb. Inhibition of Notch signalling by GSI-X inhibited the activation of T cells and affected proximal T cell signalling by regulating CD3-ζ chain expression. Inhibition of Notch signalling decreased the protein expression of CD3-ζ chain and induced expression of E3 ubiquitin ligase (GRAIL) in human αβ T cells. Apart from affecting proximal TCR signalling, Notch signalling also regulated the distal TCR signalling events. In the absence of Notch signalling, α-CD3/CD28 mAb induced activation and IFN-γ production by αβ T cells was down-modulated. The absence of Notch signalling in human αβ T cells inhibited proliferative responses despite strong signalling through TCR and IL-2 receptor. This study shows how Notch signalling cooperates with TCR signalling by regulating CD3-ζ chain expression to support proliferation and activation of human αβ T cells.     

熊去氧胆酸联合非诺贝特治疗PBC患者血清TGF-β、IFN-γ和IL-10水平变化*

目的 探讨熊去氧胆酸(UDCA)联合非诺贝特治疗原发性胆汁性胆管炎(PBC)患者血清转化生长因子-β(TGF-β)、γ-干扰素(IFN-γ)和白介素-10(IL-10)水平的变化。方法 2016年12月～2018年12月我科诊治PBC患者48例,随机将患者分为观察组(n=24)和对照组(n=24)。给予对照组患者UDCA治疗半年,给予观察组UDCA联合非诺贝特口服治疗半年。采用ELISA法检测血清TGF-β、IFN-γ和IL-10水平。使用FibroTouch行肝脏硬度测定(liver stiffness measurement, LSM)。结果 在治疗观察结束时,观察组血清谷丙转氨酶水平为(51.4±23.7)U/L,显著低于对照组【(74.9±21.2)U/L,P<0.05】,谷草转氨酶为(59.5±32.3)U/L,显著低于对照组【(81.3±35.8)U/L,P<0.05】,谷氨酰转肽酶水平为(95.7±31.8)U/L,显著低于对照组【(127.3±50.7)U/L,P<0.05】;观察组血清IFN-γ水平为(57.4±21.3)pg/mL,显著高于对照组【(39.7±23.7)pg/mL,P<0.05】,而血清TGF-β水平为(14.3±4.8)pg/mL,显著低于对照组【(23.6±3.5)pg/mL,P<0.05】;观察组血清免疫球蛋M(IgM)为(2.3±0.4)g/L,显著低于对照组【(3.1±0.9)g/L,P<0.05】, IgG水平为(11.3±1.8)g/L,显著低于对照组【(15.5±1.3)g/L,P<0.05】,IgA水平为(2.7±0.6)g/L,显著低于对照组【(3.5±0.2)g/L,P<0.05】;观察组患者LSM为(10.8±6.5)kPa,与对照组的(9.7±7.7)kPa比,无统计学差异(P>0.05)。结论 熊去氧胆酸联合非诺贝特联合治疗PBC患者可以明显改善血生化指标,可能与抑制了免疫球蛋白水平和提高了血清IFN-γ水平有关,其治疗的远期疗效还有待于观察。     

闪式提取法提取五味子醇溶性有效成分的工艺研究

目的：优化闪式提取法提取五味子醇溶性有效成分的最佳工艺。方法以五味子乙素含量为考察指标,通过单因素考察试验,首先确定影响因素的显著性及影响范围,采用正交实验设计方法,优化闪式提取五味子醇溶性有效成分的最佳工艺。结果最佳工艺条件：10倍量浓度为80％乙醇浸泡4 h,闪式提取100 s。结论闪式提取法是一种高效、快速、简单的提取方法,适合醇溶性有效成分的提取。     

PPARγ在子宫内膜异位症中的作用研究进展

现有的研究已阐明激活PPARγ具有抑制炎症反应、抗血管生成等作用,而炎症反应和血管生成均参与了子宫内膜异位症的发生发展,并且PPARγ在正常子宫内膜组织及异位子宫内膜组织均有表达。PPARγ在子宫内膜异位症中的作用受到越来越多的关注,该文就PPARγ在子宫内膜异位症中的意义作一综述。     

Thiazolidinediones under preclinical and early clinical development for the treatment of Parkinson’s disease

Introduction: Current treatment of Parkinson’s disease (PD) is limited to symptomatic dopaminergic therapy, while no interventions have been shown to slow down disease progression.

Areas covered: The following article highlights a group of PPAR-γ agonists called thiazolidinediones (TZDs), which are currently being tested for a putative disease-modifying benefit in PD, using pioglitazone as a prototypic compound. PPAR-γ is highly expressed in neurons of the substantia nigra and CNS immune cells. Preclinical data in rodent and primate support an effect of TZDs in preventing and/or arresting neurodegeneration and development of motor symptoms. Although no data on the neuroprotective effect of TZDs is currently available, a clinical trial is ongoing where the primary objective is to assess pioglitazone’s impact on the progression of PD. The trial is also evaluating the drug’s safety concerns.

Expert opinion: The efficacy data from clinical trials must be carefully weighed against the safety concerns. However, given the solid preclinical data, and since the safety data are not yet fully conclusive and limited to the diabetic population, PPAR-γ research in PD can continue with caution. Ideally, drug discovery and development efforts will lead to the identification of new compounds with reduced risk of peripheral side effects.     

蓝莓对四氯化碳所致急性肝损伤大鼠肝组织GCLCmRNA及蛋白表达的影响

目的：探讨蓝莓对四氯化碳（CCl4）所致急性肝损伤大鼠肝组织γ‐谷氨酰半胱氨酸合成酶（γ‐GCS）催化亚基（GCLC）mRNA及蛋白表达的影响。方法将50只雄性Wistar大鼠分为5组,即空白组、模型组、蓝莓汁低剂量预防组（蓝低组,10．0g／kg）、蓝莓汁高剂量预防组（蓝高组,20．0g／kg）、联苯双酯预防组（联苯双酯组,0．15g／kg）,每组10只,连续灌胃7d,1次／天,其后用CCl4复制大鼠急性肝损伤模型。光镜下观察大鼠肝脏病理学变化,全自动生化分析仪测定血清丙氨酸氨基转移酶（ALT）及天冬氨酸氨基转移酶（AST）活性,酶联免疫吸附法检测肝匀浆中丙二醛（MDA）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、还原型谷胱甘肽（GSH）活力及含量,分别采用反转录PCR（RT‐PCR）、免疫组织化学法、Westernblot检测大鼠肝组织中GCLCmRNA及蛋白表达。结果蓝低组、蓝高组和联苯双酯组大鼠肝细胞变性及坏死程度显著轻于模型组（P＜0．05）,血清ALT、AST及肝匀浆MDA均显著低于模型组（P＜0．01）,而肝匀浆CAT、SOD、GSH均显著高于模型组（P＜0．01）,肝组织GCLCmR‐NA及蛋白表达均显著高于模型组（P＜0．01）。3组相比,蓝高组与联苯双酯组效果相似,略优于蓝低组。结论蓝莓对CCl4诱导的大鼠急性肝损伤有一定预防作用,可能与上调大鼠肝脏GCLC表达,减轻氧化应激性肝损伤有关。     

泪腺炎患者血清性激素变化及其与γ-IFN、IL-4的相关性

目的 观察泪腺炎患者血清中雌二醇(estradiol,E2)、睾酮(testosterone,T)、泌乳素(prolactin,PRL)水平的变化,并探讨这种变化与血清中γ-干扰素(γ-interferon,γ-IFN)、白细胞介素4(interleukin-4,IL-4)变化的关系.方法 收集42例2013年11月至2014年10月我院确诊为泪腺炎的患者炎症期(炎症期组)与炎症缓解期(缓解期组)的血清,40例同年龄段于我院体检的正常人血清作为正常对照组,采用电化学发光法检测血清中E2、T、PRL的水平,酶联免疫吸附法测定血清中γ-IFN、IL-4水平.结果 泪腺炎患者炎症期组血清E2、PRL水平分别为(64.12±35.92) ng·L-1和(17.63±8.59) μg·L-1,显著高于缓解期组的(43.16 ±26.57)ng·L-1和(10.30±5.59) μg·L-1及正常对照组的(41.92±21.68)ng·L-1和(9.08±2.61) μg·L-1,差异均有统计学意义(均为P＜0.05);炎症期组与缓解期组血清T分别为(0.80±1.36) μg·L-1和(0.79±1.42) μg·L-1,与正常对照组的(1.76±2.49) μg·L-1相比差异均有统计学意义(均为P ＜0.05).炎症期组血清γ-IFN、IL-4水平分别为(353.12±108.36)ng·L-1和(1200.43±314.69)ng·L-1,显著高于缓解期组的(192.68±43.16)ng·L-1和(919.38±227.16)ng·L-1及正常对照组的(190.93±36.40)ng·L-1和(853.37±172.31)ng·L-1,差异均有统计学意义(均为P＜0.05).E2与γ-IFN、IL-4水平呈正相关(均为P＜0.05);T与γ-IFN、IL-4水平呈负相关(均为P＜0.05);PRL与γ-IFN、IL-4水平呈正相关(均为P＜0.05).女性患者炎症期组血清γ-IFN水平为(344.46±112.06)ng·L-1,高于男性的(292.98±71.27)ng·L-1(P＜0.05),E2/T比值为665.36,明显高于男性的13.91(P ＜0.05).结论 泪腺炎患者血清中性激素E2、PRL的升高、T的降低及E2/T比例失调可能介导Th1、Th2细胞免疫通路的平衡紊乱从而致病.女性体内高水平的雌激素可能是导致女性易患自身免疫性疾病的主要原因.