Protein kinase C mediates neural induction in Xenopus laevis

Inductive cell interactions are essential in early embryonic development, but virtually nothing is known about the molecular mechanisms involved. Recently factors resembling fibroblast growth factor and transforming growth factor-beta were shown to be involved in mesoderm induction in Xenopus laevis, suggesting that membrane receptor-mediated signal transduction is important in induction processes. Here we report direct measurements of protein kinase C (PKC) activity in uninduced ectoderm, and in neuroectoderm shortly after induction by the involuting mesoderm, in Xenopus laevis embryos. Membrane-bound PKC activity increased three to fourfold in the induced neuroectoderm while the cytosolic PKC activity was decreasing, indicating that PKC activity was translocated during neural induction. A similar time- and dose-dependent translocation of activity was seen after incubation with the PKC activator 12-O-tetradecanoyl phorbol-13-acetate, which also induced neural tissue in competent ectoderm, suggesting that PKC is involved in the response to the endogenous inducing signal during neural induction.     

Monitoring p21 mRNA expression in living cell based on molecular beacon fluorescence increasing rate

Studying the expression level of mRNA in living cells will offer tremendous opportunities for advancement in cell biology research, disease diagnostics, and drug discovery. In this paper, a molecular beacon （MB） specific for the important tumor suppressor gene p21 has been designed and synthesized. The fluorescence signal was detected in real-time after the MB entered the cytoplasm of nasopharyngeal carcinoma cells. After injecting the p21MB into nasopharyngeal carcinoma cell and p33-transfected nasopharyngeal carcinoma cell, the consistent increase of fluorescent signal intensity was detected in both cell lines, and maximum fluorescence intensity achieved in about 15 min. In about 4 min following microinjection, the fluorescence increasing rate was significantly different between these two cell lines, which indicate the different p21 mRNA expression levels. The results obtained in the real-time detection were also validated by RT-PCR. Analysis of the initial fluorescence increasing rate can efficiently reduce the side effect of enzyme and improve the accuracy in living cell mRNA detection.     

单分子成像和追踪技术在工业微生物研究中的应用

在单分子工具和技术的迅速发展过程中,单分子荧光显微镜技术脱颖而出,可以高特异性地监测荧光标记的生物分子的时空行为,成为研究生物系统的有力工具。然而,单分子成像和追踪技术在工业微生物研究及酶工程领域中的应用仍处于起步阶段。随着生物工程技术、合成生物学和代谢工程等科学技术的迅猛发展,工业微生物的应用日趋广泛。但是,菌株往往难以在整个培养期间保持最大的生产能力,导致在经济效益上不能满足产业化的需求,成为制约微生物细胞工厂发展的一个瓶颈,对酶学研究提出新的挑战。单分子成像和追踪技术具有直接、准确、实时等监测优点,可以直接在活体生物上进行研究,成功地实现了对单分子酶催化过程的实时监控,发现了多种酶的新单分子行为及反应机制。这些技术有助于各种新型酶、新特性酶、新作用方式酶的研发。本文简述单分子成像和追踪技术的主要特点（以荧光显微镜为主）,总结近年来单分子成像和追踪技术在微生物研究、酶学研究中的应用,及其在工业微生物研究中的应用现状和面临的挑战。这些技术的应用必将促进纳米生物学的发展,推动酶工程改造,提高细胞工厂的生产能力。     

Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels

It has long been assumed that a rise in cytosolic free Ca2+, [Ca2+]i, is a necessary and sufficient event for the stimulation of a variety of cellular processes. The development of a technique which allows monitoring of [Ca2+]i in small intact cells has led to a critical revision of this simple postulate. We have recently shown that in neutrophils, Ca2+-ionophore-induced elevations of [Ca2+]i, quantitatively similar to those caused by chemotatic peptides, are ineffective in stimulating cell responses, which suggests that an additional signal is required for receptor-mediated activation. Here we show that subthreshold concentrations of phorbol myristate acetate (PMA) and of a Ca2+ ionophore can quantitatively mimic the effect of a physiological agonist. However, PMA at higher concentrations can trigger NADPH-oxidase activity, exocytosis and protein phosphorylation, even when [Ca2+]i is lowered 10-20 times below the normal resting level. These results strongly suggest that activation of protein kinase C is sufficient, by itself, to induce NADPH-oxidase activation and exocytosis of secondary granules in neutrophils.     

Modulating multidrug resistance through inhibiting of protein kinase C activity by phenothiazines

In the present study, actions of phenothiazines(PTZ) in reversing multidrug resistance(MDR) and inhibiting PKC activity were investigated. It was found that the three PTZs caused 2.49, 36.58 and 75.78 fold reversal of K562/AO2 MDR cells resistant to adriamycin, respectively, while the chemosensitizer verapamil caused 40 fold reversal in the same condition, indicating that PTZ11 is a novel reversal agent of MDR and a potential chemotherapeutic reagent for tumor therapy. PKC activity analysis in the presence of PTZs showd that PTZ6 and PTZ11 inhibited rat brain protein kinase C activity in a manner of dose_dependent. The IC 50 values were (489.77±31.4) and (113±9.64) μmol/L, respectively. PTZ7 had no inhibition on PKC activity. Further study showed that PTZ11 could reduce PMA_mediated activation of PKC in a manner of dose_dependent, suggesting that PTZ11 might compete for the high_affinity phorbol ester binding site within PKC molecule. Recently, an X_ray structure of PMA in complex with PKC Cys2 activator_binding domain was solved. We therefore decided to explore the possible binding model of PTZ11 with PKC molecule using SYBYL 6.02 program. It was shown that the binding site of PTZ11 with PKC molecule partially overlapped with that of PMA, providing for the first time new data for designing PKC inhibitors and MDR reversal drugs.     

一种基于Propidium Monoazide与PCR结合的选择性检测细菌活细胞的方法

建立了一种PMA与PCR结合的细菌活细胞检测的方法,可有效抑制微生物死细胞DNA的PCR扩增,从而排除死细胞对微生物活细胞检测的影响．结果表明,PMA浓度大于3 μg/ml时可抑制死细胞DNA的PCR扩增,PMA浓度高达50 μg/ml时仍不影响活细胞DNA的PCR扩增;并且得出曝光时间大于3 min可使PMA与死细胞DNA分子共价交联,抑制其PCR扩增;浊度影响PMA交联的结果表明浊度小于10 NTU时,PMA仍能有效地抑制死细胞DNA的PCR扩增,而当浊度大于100 NTU时,PMA失去效果．     

Ia binding ligands and cAMP stimulate nuclear translocation of PKC in B lymphocytes

Altered subcellular distribution and activity of protein kinase C (PKC) is associated with transmembrane signalling in a variety of systems in which receptor occupancy leads to increased hydrolysis of polyphosphoinositides. Here we report evidence that in B lymphocytes, cyclic-cAMP-generating signal transduction pathways can activate translocation of PKC from the cytosol to the nucleus. Elevated cAMP levels and translocation of PKC to the nucleus are induced by antibodies against Ia antigens in normal B lymphocytes. Further, cAMP analogues mediate the translocation of PKC to the nucleus of these cells. These findings suggest that in physiological situations, ligation of B-lymphocyte Ia molecules by helper T cells leads to increased cAMP production which in turn causes PKC translocation to the nucleus. In view of recent observations that antibodies against Ia antigens induce differentiation of B cells, we conclude that nuclear PKC may function in the regulation of gene expression.     

丹参酮Ⅱ A通过调控PKC/CyclinD_1通路抗大鼠心肌成纤维细胞增殖作用研究

目的观察丹参酮ⅡA(TaishinoneⅡA,TSN)和蛋白激酶C(PKC)抑制剂白屈菜红碱(Chelerythrine,Chele)抗血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)诱发的心成纤维细胞(Cardiac fibroblast,CFb)增殖、细胞周期、Ⅰ型胶原纤维(CollagenⅠ)、PKC和细胞周期蛋白Cyclin D1表达的影响,阐明TSN抗CFb增殖的分子机制.方法培养的新生Wistar大鼠CFb分为对照组、AngⅡ组、Chele+AngⅡ组、Chele+AngⅡ+TSN组和AngⅡ+TSN组,胰酶消化、差速贴壁培养CFb,噻唑蓝(MTT)比色法检测细胞增殖;免疫细胞化学染色(IC)法测定Collagen I含量;流式细胞仪(FCM)检测细胞周期;免疫印迹法(WB)检测PKC和细胞周期蛋白Cyclin D1表达.结果与AngⅡ组比较,Chele和TSN组及Chele+AngⅡ+TSN组能显著降低CFb增殖率(P0.05或P0.001),降低CollagenⅠ含量(P0.05或P0.01),提高CFb G0/G1期细胞百分比,降低S期细胞百分比(P0.05或P0.01),抑制PKC和Cyclin D1蛋白表达(P0.05或P0.01).结论 TSN和Chele能显著抑制由AngⅡ诱导的CFb增殖和胶原蛋白分泌,其机制可能是通过抑制PKC-Cyclin D1传导通路实现的.     

移动变电站远程监测系统开发

基于GSM公网短信平台的远程监测技术是目前较为流行的监测技术.根据移动变电站长期处于野外无人值守的工作特点,采用西门子公司最先进的GSM通信模块TC35 i作为短信息收发核心,针对移动变电站监测系统的功能、技术特点和使用要求,在深入研究短信息收发技术、数据库技术、GSM通信模块接口电路设计技术、移动变电站状态监测技术以及电磁兼容技术的基础上,开发了基于GSM公网短信平台的移动变电站远程监测系统,现场运用表明,系统具有状态监视实时性强、信号覆盖范围广、信息传输灵活可靠、运营成本低的特点.     

基于MODBUS TCP/IP协议的海上油田发电机组网络监控系统

海上采油平台原有的发电机组检测装置因其功能单一、且无法进入监控调度级的局域网等原因而需要升级。设计了一套基于OPC通讯的多控制器、多客户机的网络监控系统,介绍了在C/S构架下MODBUS TCP/IP技术在系统中的通信原理和实现方法。以KEPServerEX作为OPC服务器,在机组检测装置与WinCC软件(OPC客户端)之间建立通信,并以安装KEPServerEX软件的工业控制计算机为C/S服务器,将各客户机和服务器相连接,MODBUS TCP/IP协议与OPC技术的应用,提高了通信的速度及系统的扩展性。实际运行表明,基于MODBUS TCP/IP和OPC技术的多WinCC组态与多发电机组检测装置间通信实时性与稳定性良好。     

振动信号分析用于出砂监测方法有效性研究

 海上稠油油田的适度出砂开采技术需要一种有效的实时出砂监测系统用以监测出砂信号,目前使用的出砂声测法和ER法都存在不同的问题。声测法目前主要用于气井和稀油油井出砂检测中,而ER法则存在寿命短、监测延迟等问题。因此,提出了可以利用流体中的砂粒撞击管壁产生的振动信号作为出砂监测的新思路。为了验证这一方法的有效性,在实验室内建立了一套用于模拟油井出砂的实验装置,系统中主要采用压电式加速度传感器接受砂粒撞击的振动信号,后期采用滤波、放大等动态信号的处理方法来处理;实验中利用高粘度的齿轮油模拟稠油流体,用柱塞泵、加热搅拌器来实现油井生产的出砂状况。实验采用60目砂样石英砂粒,室温下粘度为130mPa·s的稠油,实验过程中温度从室温逐渐增加,每5℃一个点,到70℃,含砂量从无砂加到0.8%,实验过程中通过不断搅拌保持砂粒在流体中的悬浮和均匀。通过对采集到的数据进行功率谱分析,试验结果表明,将振动信号用于出砂监测有效性很好。     

甲基乙二醛诱导牙周膜成纤维细胞基因表达与分析

运用基因芯片研究甲基乙二醛诱导人牙周膜成纤维细胞基因表达谱的变化。原代培养人牙周膜成纤维细胞,诱导组以终质量浓度为0.1 g/L的甲基乙二醛刺激培养细胞,对照组不含甲基乙二醛。24 h后收获细胞,提取mRNA,逆转录cDNA时用Cy3和Cy5荧光染料标记,制备成cDNA探针,与表达谱芯片进行杂交、扫描和分析。芯片检测结果用实时定量聚合酶链反应验证和生物信息学分析。结果共有18条基因显著差异表达,其中上调基因有11条,下调基因有7条,差异性表达的基因按功能可分为程序性细胞死亡、信号转导、细胞因子、代谢酶类、载体蛋白和未知基因等。与程序性细胞死亡、信号转导和细胞因子相关基因的差异表达可能是甲基乙二醛通过线粒体信号通路,诱导人牙周膜成纤维程序性细胞死亡,破坏牙周组织增生,从而导致牙周病发生的机制。     

表面等离子体共振免疫传感的放大检测研究

表面等离子体共振免疫传感器是一类相对新型的免疫检测技术。将链霉亲和素-生物素系统用于表面等离子体共振免疫传感的信号放大,实时检测了人免疫球蛋白G(hIgG)的蛋白浓度。发生免疫反应的传感片和生物素化抗体反应后,传感片表面的一层生物素分子随后与链霉亲和素-生物素化抗体复合物中的链霉亲和素的活性位点发生亲和反应,从而使传感片表面特异健合的物质质量显著增加,大大提高了免疫检测的灵敏度和检测限。免疫反应经放大后,可检测0.005～10μg/mL浓度区间内的hIgG。     

NO对新生大鼠心肌细胞PKC活性的影响

利用培养新生大鼠心肌细胞，检测NO前体L-精氨酸(L-arginine,L-Arg)和NO供体硝普钠(sodium nitroprusside,SNP)对PKC活性的影响，并探讨内、外源性NO在PKC激动剂佛波指(phorbol 12-myristate 13-acetate,PMA)激活PKC中的作用.实验结果表明：培养基中加入L-Arg，PKC活性呈剂量依赖性降低；用L-Arg进行预处理，30 min后加入PMA，PKC活性明显降低，与单纯PMA组相比有显著差异；NOS抑制剂L-NAME本身对基础状态PKC活性无明显影响，但可阻断L-Arg对上述2个效应的影响；培养液中加入NO供体SNP，PKC活性呈剂量依赖性降低；用SNP预处理心肌细胞，5 min后加入PMA，PKC活性与单纯PMA组相比有显著性差异.以上结果表明，内、外源性NO均具有剂量依赖性抑制PKC活性的作用，PKC可能是NO对心肌细胞作用的胞内信号传导通路的关键部位或重要信号分子之一；L-Arg通过NOS先生成NO，NO再对PKC起抑制作用.     

基于软件无线电的无线信号实时监测系统的若干关键技术

该文介绍了一种基于软件无线电技术的无线信号实时分析系统．该系统采用了改进的快速傅立叶变换算法和多次采样技术,提高了信号的实时处理效率和输出信噪比,从而改善系统信号处理的实时性和可靠性。     

Single-molecule fluorescence imaging of membrane-bound proteins for studies of cell signal transduction

Fluorescence imaging of single molecules is becoming a powerful tool to examine biological processes at the molecular level.Using total internal reflection fluorescence microscopy (TIRFM),it has been possible to study the dynamic behavior of single molecules on living cell membranes.Herein,we briefly review the application of TIRFM-based single-molecule imaging in studies of membrane receptors involved in signal transduction.Furthermore,we discuss several examples of our own research on growth factor receptors,including TGF-β receptors,HER2,and EGFR,and speculate possible applications of this technique to investigate other cellular events occurring on or near the plasma-membrane.     

Quantitative detection of ING1 mRNA under different gene regulation based on molecular beacon

As an important tumor suppressor, normal expres- sion of ING1 is associated with the maintenance of cell normal physiological functions. Its low expression of- ten causes aberrant events of cell survive, growth and differentiation[1] which results in loss…     

Potentiation of synaptic transmission in the hippocampus by phorbol esters

Protein kinase C (PKC), a calcium-dependent phospholipid-sensitive kinase which is selectively activated by phorbol esters, is thought to play an important role in several cellular processes. In mammalian brain PKC is present in high concentrations and has been shown to phosphorylate several substrate phosphoproteins, one of which may be involved in the generation of long-term potentiation (LTP), a long-lasting increase in synaptic efficacy evoked by brief, high-frequency stimulation. Since the hippocampus contains one of the brain's highest levels of binding sites for phorbol esters and is the site where LTP has been most thoroughly characterized, we examined the effects of phorbol esters on hippocampal synaptic transmission and LTP. We found that phorbol esters profoundly potentiate excitatory synaptic transmission in the hippocampus in a manner that appears indistinguishable from LTP. Furthermore, after maximal synaptic enhancement by phorbol esters, LTP can no longer be elicited. Although the site of synaptic enhancement during LTP is not clearly established, phorbol esters appear to potentiate synaptic transmission by acting primarily at a presynaptic locus since changes in the postsynaptic responses to the putative transmitter, glutamate, cannot account for the increased synaptic responses induced by phorbol esters. These findings, in conjunction with previous biochemical studies, raise the possibility that, in mammalian brain, PKC plays a role in controlling the release of neurotransmitter and may be involved in the generation of LTP.     

肿瘤坏死因子(TNF)的分子结构和生物学活性

TNF最初被发现是因为它显示了抗癌活性,现已判定它是免疫细胞接受某些特定的刺激因子所产生的多功能细胞因子,它参与了炎症和免疫反应以及细胞生长或抑制的各种蛋白质因子中的一族,本文讨论了TNF的分子结构和生物学活性及两者之间的关系。