SH2A 基因对细胞信号转导的影响及其亚细胞定位

目的 研究Src同源域2(src homology 2，SH2)A基因在细胞信号转导中的作用并进行亚细胞定位。方法 通过RT—PCR方法扩增SH2A cDNA编码序列，构建真核重组表达载体pcDNA3．1-SH2A，利用脂质体转染肝癌Bel7402细胞、COS7细胞，检测蛋白酶C(protein kinaseC，PKC)、酪氨酸蛋白激酶(tyrosine protein kinase，TPK)、丝裂原激活蛋白激酶(mitogen activated protein kinase，MAPK)活性的改变；另构建pEGEP—SH2A，转染同前，荧光显微镜观察荧光定位。结果 测序结果显示真核重组表达载体pcDNA3．1-SH2A及pEGFP—SH2A中均含有SH2AcDNA编码序列；肝癌Bel7402细胞、COS7细胞转染pcDNA3．1-SH2A后，胞浆PKC的活性下降了40％左右，MAPK和TPK活性未见明显改变。荧光显微镜观察发现SH2A基因在细胞质中表达。结论 SH2A基因编码蛋白在PKC信号转导通路中起抑制作用；SH2A基因编码蛋白定位于细胞质。     

A Parametric Approach to Measuring Cerebral Blood Flow Autoregulation from Spontaneous Variations in Blood Pressure

Autoregulation maintains cerebral blood flow (CBF) almost constant in the face of changes in arterial blood pressure (ABP). Tests for impairment of this process using only spontaneous fluctuations in ABP, without provoking large variations, are of great clinical interest, and a range of different approaches have previously been applied. Extending earlier work based on linear filters, we propose a simple parametric method using a first order finite impulse response filter. We evaluate the method on ABP and CBF velocity [(CBFV), from trancranial Doppler ultrasound] signals collected in 60 patients with stenosis or occlusion of the carotid arteries. Data were collected during the inspiration of ambient air, a 5% CO2/air mixture, and finally the return to ambient air. Equivalent data were collected in 15 normal subjects. The filters estimated from the data segments with constant inspiratory pCO2 showed the expected high-pass characteristic, which was reduced during hypercapnia and also in patients. Highly significant correlation between the filter parameters and cerebrovascular reactivity (percent increase in CBFV per unit change in end-tidal pCO2) gives further evidence that the filters reflect autoregulation. The method allows simple parametrization of the dynamic autoregulatory responses in CBFV, and the analysis of short (1 min) data segments. © 2001 Biomedical Engineering Society. PAC01: 8719Uv, 4762+q, 4380Qf     

STAT3 couples with 14-3-3σ to regulate BCR signaling,B-cell differentiation,and IgE production

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上皮间质转化(EMT)及其分子机制

上皮细胞受到细胞外因子刺激后向间质细胞转化的现象与肿瘤的浸润转移密切相关。在此过程中上皮细胞的极性丧失，迁移和运动能力增强，同时上皮表型丢失而逐渐获得间质表型。参与这一过程的细胞内信号转导途径有受体酪氨酸激酶Ras-MAPK途径，Src激酶，Rho家族激酶，PI3K/AKT途径，Wnt信号通路，转录因子等。     

Signal-dependent wavelets for electromyogram classification

In the study, an efficient method to perform supervised classification of surface electromyogram (EMG) signals is proposed. The method is based on the choice of a relevant representation space and its optimisation with respect to a training set. As EMG signals are the summation of compact-support waveforms (the motor unit action potentials), a natural tool for their representation is the discrete dyadic wavelet transform. The feature space was thus built from the marginals of a discrete wavelet decomposition. The mother wavelet was designed to minimise the probability of classification error estimated on the learning set (supervised classification). As a representative example, the method was applied to simulate surface EMG signals generated by motor units with different degrees of short-term synchronisation. The proposed approach was able to distinguish surface EMG signals with degrees of synchronisation that differed by 10%, with a misclassification rate of 8%. The performance of a spectral-based classification (error rate approximately 33%) and of the classification with Daubechies wavelet (21%) was significantly poorer than with the proposed wavelet optimisation. The method can be used for a number of different application fields of surface EMG classification, as the feature space is adapted to the characteristics of the signal that discriminate between classes. An erratum to this article is available at .     

细胞穿透肽PEP-1介导增强型绿色荧光蛋白在小鼠体内跨膜转导

目的 研究细胞穿透肽PEP-1介导的大分子物质在小鼠体内的跨膜转导能力.方法 用基因工程的方法制备并纯化增强型绿色荧光蛋白EGFP和PEP-1-EGFP融合蛋白,分别将500 μg的EGFP蛋白和PEP-1-EGFP融合蛋白通过尾静脉注射入昆明小鼠体内,2 h后麻醉小鼠,PBS充分灌流并取心、脑、肝、脾、肾快速冷冻切片后立即置荧光显微镜下观察.结果 2 h后PEP-1-EGFP融合蛋白处理的小鼠大脑、心肌、肝、脾和肾组织里出现均一的明亮绿色荧光,而EGFP蛋白处理的小鼠各脏器内均未见到绿色荧光.结论 细胞穿透肽PEP-1能携带增强型绿色荧光蛋白穿透小鼠细胞膜并分布于心、脑、肝、脾、肾组织内,为将来用PEP-1介导各种大分子药物跨膜转导进行各种疾病的蛋白治疗提供实验依据.     

尿激酶型纤溶酶原激活物系统对骨巨细胞瘤细胞基质金属蛋白酶-2和组织基质金属蛋白酶抑制物-3表达的影响

目的:研究尿激酶型纤溶酶原激活物(uPA)及其受体(uPAR)信号转导对骨巨细胞瘤基质金属蛋白酶-2(MMP-2)和金属蛋白酶组织抑制物-3(TIMP-3)的调节。方法:用免疫组化检测骨巨细胞瘤组织中uPAR、MMP-2和TIMP-3的表达。用免疫共沉淀法检测uPA对瘤细胞信号转导通路的p44蛋白磷酸化水平。用蛋白印迹法检测用uPA和uPAR抗体处理后瘤细胞MMP-2和TIMP-3蛋白表达。结果:(1)uPAR主要表达在部分单核基质细胞和一些多核巨细胞的胞膜上;(2)MMP-2主要表达在瘤细胞的胞浆,在多核巨细胞,其表达有明显的极向性;(3)在骨巨细胞瘤组织TIMP-3表达量低于MMP-2,在多核巨细胞也显示极向性表达;(4)将uPA-ATF加入培养的骨巨细胞瘤细胞后,细胞信号通路上的p44蛋白磷酸化水平明显增高。用uPAR抗体处理后,细胞p44蛋白磷酸化水平明显降低。说明uPA-ATF参与细胞信号转导,而且受uPAR拮抗剂的影响;(5)uPA-ATF信号通路上调MMP-2和TIMP-3的表达,而uPAR抗体则下调MMP-2和TIMP-3的表达。结论:本实验首次直接证明uPA-ATF通过信号转导能调节MMP-2和TIMP-3的表达,而后者则在骨巨细胞瘤局部骨质吸收中起重要作用。     

T cell receptor induced intracellular redistribution of type I protein kinase A

The productive activation of CD4(+) T lymphocytes, leading to proliferation and cytokine secretion, requires precise temporal regulation of intracellular cyclic AMP concentrations. The major effector molecule activated by cyclic AMP in mammalian cells is the cyclic AMP-dependent protein kinase A (PKA). The type I PKA isozyme mediates the inhibitory effects of cyclic AMP on T-cell activation. Using laser scanning confocal microscopy, we demonstrated that the regulation of PKA type I activity involves spatial redistribution of PKA type I molecules following T-cell receptor (TCR) stimulation. In resting T cells, PKA type I was located in membrane proximal regions and distributed equally across the cell. Shortly after antigen engagement, T cells and antigen-presenting cells formed an area of intense contact, known as the immunological synapse. TCR concentrated at the synapse, whereas PKA type I molecules redistributed to the opposite cell pole within 10 min after T-cell stimulation. Type I PKA redistribution was solely dependent on TCR signalling, because we observed the same temporal and spatial distribution after antibody-mediated cross-linking of the TCR-associated CD3 complex. Segregation of TCR and PKA type I molecules was maintained for at least 20 min. Thirty minutes after stimulation, PKA type I partially colocalized with the TCR. After 60 min, PKA type I distribution again approached the resting state. Considering that initial TCR signals lead to increases in intracellular cyclic AMP, PKA type I molecules may be targeted towards localized cyclic AMP accumulations or transported away from these areas, depending on the requirements of the cellular response.     

Physiological mechanisms of TRPC activation

TRPC (canonical transient receptor potential) channels are vertebrate homologs of the Drosophila photoreceptor channel, TRP. Considerable research has been brought to bear on the seven members of this family, especially with regard to their possible role in calcium entry. Unfortunately, the current literature presents a confusing picture, with different laboratories producing widely differing results and interpretations. It appears that ectopically expressed TRPC channels can be activated by phospholipase C products (generally, diacylglycerols), by stimulation of trafficking to the plasma membrane, or by depletion of intracellular Ca²⁺ stores. Here, I discuss the possibility that these diverse experimental findings arise because TRPC channels can, under both experimental as well as physiological conditions, be activated in three distinct ways, possibly depending on their subunit composition and/or signaling complex environment. The TRPCs may be unique among ion-channel subunit families in being able to participate in the assembly and function of multiple types of physiologically important ion channels.