Exendin 4高活性短肽模拟物的筛选及生物活性#br#

以细胞表面胰高血糖素样肽(GLP-1)受体为靶标, 利用噬菌体筛选技术, 通过Exendin-4竞争性洗脱筛选GLP-1受体激动剂, 得到了12肽模拟物EPA. 细胞增殖实验结果表明： EPA和Exendin-4均可促进胰岛β细胞增殖活性, 在40~200 μmol/L内, EPA比Exendin-4的活性高5%~25%; 在100 mmol/L高浓度葡萄糖毒性下, EPA通过抑制bax基因和上调bcl 2基因表达及抑制Caspase 3活性来抑制胰岛β细胞的凋亡; EPA是具有高活性的Exendin-4短肽模拟物, 可通过bcl-2/bax…Caspase 3信号通路抑制线粒体的凋亡.     

Na(+)/H(+ ) exchanger regulatory factor 2 directs parathyroid hormone 1 receptor signalling

The parathyroid hormone 1 receptor (PTH1R) is a class II G-protein-coupled receptor. PTH1R agonists include both PTH, a hormone that regulates blood calcium and phosphate, and PTH-related protein (PTHrP), a paracrine/autocrine factor that is essential for development, particularly of the skeleton. Adenylyl cyclase activation is thought to be responsible for most cellular responses to PTH and PTHrP, although many actions appear to be independent of adenylyl cyclase. Here we show that the PTH1R binds to Na(+)/H(+) exchanger regulatory factors (NHERF) 1 and 2 through a PDZ-domain interaction in vitro and in PTH target cells. NHERF2 simultaneously binds phospholipase C beta 1 and an atypical, carboxyl-terminal PDZ consensus motif, ETVM, of the PTH1R through PDZ1 and PDZ2, respectively. PTH treatment of cells that express the NHERF2 PTH1R complex markedly activates phospholipase C beta and inhibits adenylyl cyclase through stimulation of inhibitory G proteins (G(i/o) proteins). NHERF-mediated assembly of PTH1R and phospholipase C beta is a unique mechanism to regulate PTH signalling in cells and membranes of polarized cells that express NHERF, which may account for many tissue- and cell-specific actions of PTH/PTHrP and may also be relevant to signalling by many G-protein-coupled receptors.     

十拷贝串联重复laGLP-1基因植物表达载体的构建及对黄瓜遗传转化的研究

天然胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)是含有30个氨基酸的短肽激素,对2型糖尿病具有特殊疗效.以本实验室克隆的长效GLP-1(long acting GLP-1,laGLP-1)基因为基础,构建了十拷贝串联重复laGLP-1的无抗生素选择标记的植物表达载体pX6-laGLP-1,采用农杆菌介导法对黄瓜自交系2M1进行转化,通过PCR及Southern杂交检测分析,最后获得了4株稳定整合十拷贝串联重复laGLP-1的转基因植株.该遗传体系的建立为研发糖尿病食疗型转基因植物或用其生产治疗糖尿病的口服药物奠定了基础.     

重组大肠杆菌高密度发酵表达胰高血糖素样肽-1衍生物(GP62)的培养基优化

对重组E.coli BL21 (DE3)高密度表达胰高血糖素样肽-1衍生物(GP62)的发酵培养基进行了优化.通过单因素实验与正交实验相结合的方法,依次对基础培养基,发酵培养基中碳源、氮源、无机盐等成份进行优化.在2YT培养基基础上,获得优化的发酵培养基2YT-GP62.在5L全自动发酵罐中利用终浓度0.5 mmol/...     

Movement of 'gating charge' is coupled to ligand binding in a G-protein-coupled receptor

Activation by agonist binding of G-protein-coupled receptors (GPCRs) controls most signal transduction processes. Although these receptors span the cell membrane, they are not considered to be voltage sensitive. Recently it was shown that both the activity of GPCRs and their affinity towards agonists are regulated by membrane potential. However, it remains unclear whether GPCRs intrinsically respond to changes in membrane potential. Here we show that two prototypical GPCRs, the m2 and m1 muscarinic receptors (m2R and m1R), display charge-movement-associated currents analogous to 'gating currents' of voltage-gated channels. The gating charge-voltage relationship of m2R correlates well with the voltage dependence of the affinity of the receptor for acetylcholine. The loop that couples m2R and m1R to their G protein has a crucial function in coupling voltage sensing to agonist-binding affinity. Our data strongly indicate that GPCRs serve as sensors for both transmembrane potential and external chemical signals.     

Calcitonin gene-related peptide regulates calcium current in heart muscle

The influx of Ca2+ due to the transmembrane calcium current, ICa, has a fundamental role in cardiac pacemaker activity, in the action potential plateau and in excitation-contraction coupling. Both sympathetic and parasympathetic neurotransmitters can modulate ICa. Recent studies indicate that in both the cardiovascular and the central nervous systems, nerve varicosities exist that contain a novel non-adrenergic, non-cholinergic peptide--calcitonin gene-related peptide (CGRP). Although CGRP is known to exert strong positive inotropic and chronotropic effects, as well as to cause vasodilation, very little is known about the ionic mechanisms of these effects. Here we report that CGRP dramatically increases ICa in single heart cells. Although this CGRP-induced increase in ICa resembles the effect of beta-adrenergic agonists, our results demonstrate some significant differences between the effects of CGRP and these agonists: (1) the increase due to CGRP cannot be blocked by beta-adrenergic antagonists; (2) the CGRP-induced effect is transient; and, (3) CGRP can inhibit isoproterenol-stimulated ICa. Our results provide the first electrophysiological evidence that CGRP can significantly modulate ICa in the heart, and suggest a new additional mechanism for the neurogenic control of cardiac function.     

Distinct molecular mechanism for initiating TRAF6 signalling

Tumour-necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is the only TRAF family member that participates in signal transduction of both the TNF receptor (TNFR) superfamily and the interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily; it is important for adaptive immunity, innate immunity and bone homeostasis. Here we report crystal structures of TRAF6, alone and in complex with TRAF6-binding peptides from CD40 and TRANCE-R (also known as RANK), members of the TNFR superfamily, to gain insight into the mechanism by which TRAF6 mediates several signalling cascades. A 40 degrees difference in the directions of the bound peptides in TRAF6 and TRAF2 shows that there are marked structural differences between receptor recognition by TRAF6 and other TRAFs. The structural determinant of the petide TRAF6 interaction reveals a Pro-X-Glu-X-X-(aromatic/acidic residue) TRAF6-binding motif, which is present not only in CD40 and TRANCE-R but also in the three IRAK adapter kinases for IL-1R/TLR signalling. Cell-permeable peptides with the TRAF6-binding motif inhibit TRAF6 signalling, which indicates their potential as therapeutic modulators. Our studies identify a universal mechanism by which TRAF6 regulates several signalling cascades in adaptive immunity, innate immunity and bone homeostasis.     

Opposite effects of cyclic GMP and cyclic AMP on Ca2+ current in single heart cells

The slow inward Ca2+ current, ICa, is fundamental in the initiation of cardiac contraction and neurohormonal regulation of cardiac function. It is increased by beta-adrenergic agonists, which stimulate synthesis of cyclic AMP (cAMP) and cAMP-dependent phosphorylation. The neurotransmitter acetylcholine reduces ICa by an unknown mechanism. There is strong evidence that acetylcholine reduces ICa by decreasing adenylate cyclase activity, but cGMP has also been implicated as ACh stimulates cGMP accumulation and activates cGMP-dependent protein kinase. Application of cGMP decreases contractile force, decreases Ca flux, shortens the duration of action potentials and inhibits Ca-dependent action potentials. Other studies, however, have concluded that cGMP levels do not correlate with contractile force and that cGMP has no effect on ICa. We have therefore examined the effects of intracellular perfusion of cGMP on ICa using isolated, voltage-clamped cells from frog ventricle. We find that cGMP has negligible effects on basal ICa, but greatly decreases the ICa that had been elevated by beta-adrenergic agonists or by intracellular perfusion with cAMP. The decrease of ICa is mediated by cAMP hydrolysis via a cGMP-stimulated cyclic nucleotide phosphodiesterase.     

补肾清肝疏脾法对 2 型糖尿病 SD 大鼠模型肠道菌群改善及 MAPK 信号通路的影响

摘要:目的 本研究拟评估补肾清肝疏脾法对 2 型糖尿病 SD 大鼠模型的治疗作用及其对 MAPK 信号通路的影响。 方法 选用 2 型糖尿病 SD 大鼠模型,分为模型对照组( 对照组,n = 8) 和补肾清肝疏脾法组( 实验组,n = 8) 。实验组大鼠采用蒸馏水制备的中药方剂,对照组和正常饲养组( 空白组,n = 8) 使用 无 菌 水,连 续 灌 胃 6 个 月,然后采集样品,进行肠道菌群、胰高血糖素样肽-1( GLP -1) 、酪酪 肽 ( PYY) 含 量、体 质 量、空 腹 血 糖 ( FBG) 、血 脂及胰岛素分泌指数的分析。 结果 对照组大鼠 肠 道 内 双 歧 杆 菌 和 乳 酸 杆 菌 显 著 降 低、而 粪 肠 球 菌 及 大 肠 杆菌有明显的增加。 使用补肾清肝疏 脾 法 对 大 鼠 进 行 治 疗 后, Western bolt 结 果 显 示,糖 尿 病 模 型 大 鼠 肠 道 的GLP -1 含量较治疗前有显 著 性 差 异。 ELISA 结 果 显 示,空 白 组 与 对 照 组 大 鼠 相 比, PYY 含 量 未 见 显 著 性 变化;而实验组大鼠其含量增加显 著。 实 验 组 大 鼠 的 血 糖、血 脂、 HOMA-β、体 质 量 与 对 照 组 相 比,在 3 个 月、6个月均有显著性差异。 结论 补肾清肝疏脾法可控制糖尿病的发展,其作用 机 制 可 能 是 通 过 改 善 肠 道 菌 群、GLP -1 和 PYY 含量进行的。     

新烟碱型乙酰胆碱受体吡啶基醚类配体的3维定量构效关系

采用比较分子力场分析方法，对一系列与烟碱型乙酰胆碱受体作用的吡啶基醚类配体进行构效关系分析，建立了这类化合物的3维构效模型．其交叉验证回归系数Rcv^2、非交叉验证回归系数Rncv^2和标准偏差s分别为0．744，0．973和0．256．结论说明该系列化合物的分子立体场和静电场的分布与生物活性之间有良好的相关性．该模型对训练集分子的活性预测结果较好，表明该力场模型有一定的预测能力，可用来指导设计新的烟碱型乙酰胆碱受体配体．     

Signal transduction pathways mediated by colony stimulating factor-1 receptor

Colony-stimulating factor-1 (CSF-1), which is necessary for cell proliferation and differentiation, regulates both immediate and delayed early responses throughout G1 phase. The binding of CSF-1 to its receptor (CSF-1R) triggers phosphorylation of the receptor and its intrinsic tyrosine kinase. The activated receptor binds directly to cytoplasmic effector proteins, which induce multiple-signal transduction pathways. CSF-1 can induce the c-myc gene expression via Ras and Ets-related proteins. The expression of c-fos/jun family genes is also targeted following the activation of Ras. CSF-1R activates STAT1 and STAT3 to participate in signaling, but JAKs do not appear to contribute to signaling by CSF-1R. CSF-1R activates PI3-kinase, and PI3-ki can interact with downstream proteins by the MAPKK-related pathway independent of Ras/Raf. PC-PLC can enforce signaling in response to CSF-1. Furthermore, the turnover and dephosphorylation by the phosphatase SHPTP1 of CSF-1R are the major mechanism in the negative regulation of signaling by CSF-1R     

A system for screening agonists targeting β2-adrenoceptor from Chinese medicinal herbs

In order to develop a model for screening the agonists of human β₂-adrenoceptor from Chinese medicinal herbs extracts, we used a cell-based functional assay based on a common G protein-coupled receptor (GPCR) regulation mechanism and destabilized enhanced green fluorescent protein (d₂EGFP) reporter gene technique. The positive cell clone was confirmed by real-time polymerase chain reaction (PCR) and imaging analysis. To assess the value of this model, we screened over 2000 high performance liquid chromatography (HPLC)-fractionated samples from the ethanol extracts of Chinese medicinal herbs. Six fractions (isolated from Panax japonicus, Veratrum nigrum, Phellodendron amurense, Fructus Aurantii Immaturus, Chaenomeles speciosa, and Dictamnus dasycarpus) showed significant effects on active reporter gene expression, three of which (isolated from Phellodendron amurense, Fructus Aurantii Immaturus, and Chaenomeles speciosa) were selected for further concentration response analysis and the half maximal effective concentration (EC_{1/2 max}) values were 4.2, 2.7, and 4.8 µg/ml, respectively. Therefore, this reporter gene assay was suitable for screening β₂-adrenoceptor agonists. The results suggest that the six herbal extracts are the possible agonists of β₂-adrenoceptor.     

A new long-acting GLP-1 derivative KTP ameliorates hyperglycemia and dyslipidemia and improves pancreas and fatty liver in db/db mice

Glucagon-like peptide-1 (GLP-1) is a polypeptide incretin hormone that glucose-dependently promotes the secretion and synthesis of insulin. However, its half-life is extremely short. To enhance its half-life, we developed a long-acting GLP-1 derivative KTP with controlled release, designed on the basis of another GLP-1 derivative GP62. The kinetics and bioactivity of KTP were evaluated in Sprague Dawley (SD) rats. Long-term treatment of KTP was performed in db/db mice. Mice were treated twice daily with subcutaneous injections of KTP (1.2 mg/kg body weight), Exendin-4 (0.1 mg/kg body weight) or vehicle (phosphate buffered saline (PBS), pH 7.4) for 60 d. KTP had a longer half-life, as well as further increasing the secretion and production of insulin and reducing blood glucose concentrations, than GP62. Long-term treatment with KTP also induced anorexia, decreased water and food intake, decreased weight gain, improved blood glucose and blood lipid and ameliorated pancreatic damage and fatty liver in db/db mice.     

5-HT 6受体拮抗剂三维定量构效关系研究

为指导合成高效的5-HT6受体拮抗剂,采用比较分子场分析(CoMFA)和比较相似性指数分析(CoMSIA)对143个5-HT6受体拮抗剂数据进行了三维定量构效关系(3D-QSAR)研究,分别得到了具有良好可靠性和预测能力的CoMFA(Q2=0.513,R2ncv=0.864,R2pre=0.731)和CoMSIA模型(Q2=0.515,R2ncv=0.844,R2pre=0.777).由模型的等势线图分析,可得如下结论:大体积及/或电负性较大的R2取代基、疏水性R3和疏水性苯环R1位取代基、可作氢键受体的R2取代基、可作氢键供体的R3取代基有助于增大活性.这些结论能够更好地帮助理解5-HT6受体拮抗剂的抑制机理,并为今后的药物设计与合成提供新思路.     

Dielectric relaxation and conduction mechanism in Aurivillius ceramic Bi5Ti3FeO15

For this study, we synthesized Aurivillius Bi5Ti3FeO15 ceramic using the generic solid-state reaction route and then performed room-temperature X-ray diffraction to confirm that the compound had a single phase with no impurities. The surface morphology of the prepared sample was observed to contain microstructural grains approximately 0.2–2 μm in size. The dielectric properties of the sample were determ-ined as a function of frequency in a range of approximately 100 Hz to 1 MHz at various temperatures (303 K ≤ T ≤ 773 K). Nyquist plots of the impedance data were found to exhibit a semi-circular arc in the high-temperature region, which is explained by the equivalent electrical circuit (R1C1)(R2QC2), where R1 and R2 represent the resistances associated with the grains and grain boundaries, respectively, C1 and C2 are the re-spective capacitances, and Q is the constant phase element (CPE), which accounts for non-Debye type of behavior. Our results indicate that both the resistance and capacitance of the grain boundaries are more prominent than those of the grains. The alternating current (ac) conductiv-ity data were analyzed based on the Jonscher universal power law, which indicated that the conduction process is dominated by the hopping mechanism. The calculated activation energies of the relaxation and conduction processes were very similar (0.32 to 0.53 eV), from which we conclude that the same type of charge carriers are involved in both processes.     

HO2 + ClO气相反应机理及主通道速率常数的理论研究

本文采用CCSD(T)/aug-cc-pVTZ//B3LYP/6-311+G(2df,2p)方法构建了HO_2+ClO反应体系的单、三重态反应势能剖面,并对该反应主通道的速率常数进行了计算研究.结果表明,HO_2+ClO反应中存在4条抽氢通道R1(HOCl+~1O_2)、R2(HOCl+~3O_2)、R3(HCl+~1O_3)和R4(HCl+~3O_3)以及2条抽氧通道R5(OOCl+HO)和R6(OClO+HO),其中抽氢通道R2(HOCl+~3O_2)和R3(HCl+1O3)的能垒比其它四个通道的能垒降低了9.08~42.90kcal·mol-1,是标题反应的优势通道.采用传统过渡态理论并结合Wigner校正对优势通道R2(HOCl+~3O_2)和R3(HCl+~1O_3)在240~425K范围内的速率常数进行了计算.计算结果表明,通道R2(HOCl+3 O2)的速率常数比R3(HCl+~1O_3)的对应值大了3~5个数量级,表明标题反应的速率主要取决于通道R2(HOCl+~3O_2).此外在298 K时,通道R2(HOCl+3O2)的速率常数为2.76×10-15 cm3·molecule-1·s-1,与实验值较为吻合.