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161.
162.
Accumulating evidence implicates nicotinic acid adenine dinucleotide phosphate (NAADP) in the control of Ca2+-dependent functions. Little, however, is known concerning its role in the vascular endothelium, a major regulator of blood pressure. Here, we show that NAADP acetoxymethyl ester (NAADP-AM), a cell-permeant NAADP analog, increases cytosolic Ca2+ concentration in aortic endothelial cells. We demonstrate that these signals and those evoked by acetylcholine are blocked by disrupting acidic organelles with bafilomycin A1. In contrast, Ca2+ signals in response to thrombin are only partially inhibited by bafilomycin A1 treatment, and those to ATP were insensitive, suggesting that recruitment of acidic stores is agonist-specific. We further show that NAADP-evoked Ca2+ signals hyperpolarize endothelial cells and generate NO. Additionally, we demonstrate that NAADP dilates aortic rings in an endothelium- and NO-dependent manner. Finally, we show that intravenous administration of NAADP-AM into anesthetized rats decreases mean arterial pressure. Our data extend the actions of NAADP to the endothelium both in vitro and in vivo, pointing to a previously unrecognized role for this messenger in controlling blood pressure.  相似文献   
163.
Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction.  相似文献   
164.
Bulk solution assays have shown that the isolated CARMIL homology 3 (CAH3) domain from mouse and Acanthamoeba CARMIL rapidly and potently restores actin polymerization when added to actin filaments previously capped with capping protein (CP). To demonstrate this putative uncapping activity directly, we used total internal reflection microscopy to observe single, CP-capped actin filaments before and after the addition of the CAH3 domain from mouse CARMIL-1 (mCAH3). The addition of mCAH3 rapidly restored the polymerization of individual capped filaments, consistent with uncapping. To verify uncapping, filaments were capped with recombinant mouse CP tagged with monomeric green fluorescent protein (mGFP-CP). Restoration of polymerization upon the addition of mCAH3 was immediately preceded by the complete dissociation of mGFP-CP from the filament end, confirming the CAH3-driven uncapping mechanism. Quantitative analyses showed that the percentage of capped filaments that uncapped increased as the concentration of mCAH3 was increased, reaching a maximum of ∼90% at ∼250 nm mCAH3. Moreover, the time interval between mCAH3 addition and uncapping decreased as the concentration of mCAH3 increased, with the half-time of CP at the barbed end decreasing from ∼30 min without mCAH3 to ∼10 s with a saturating amount of mCAH3. Finally, using mCAH3 tagged with mGFP, we obtained direct evidence that the complex of CP and mCAH3 has a small but measurable affinity for the barbed end, as inferred from previous studies and kinetic modeling. We conclude that the isolated CAH3 domain of CARMIL (and presumably the intact molecule as well) possesses the ability to uncap CP-capped actin filaments.  相似文献   
165.
Palmitoylation represents a common motif for anchorage of cytosolic proteins to the plasma membrane. Being reversible, it allows for controlled exchange between cytosolic and plasma membrane-bound subpopulations. In this study, we present a live cell single molecule approach for quantifying the exchange kinetics of plasma membrane and cytosolic populations of fluorescently labeled Lck, the key Src family kinase involved in early T cell signaling. Total internal reflection (TIR) fluorescence microscopy was employed for confining the analysis to membrane-proximal molecules. Upon photobleaching Lck-YFP in TIR configuration, fluorescence recovery proceeds first via the cytosol outside of the evanescent field, so that in the early phase fluorescence signal arises predominantly from membrane-proximal cytosolic Lck. The diffusion constant of each molecule allowed us to distinguish whether the molecule has already associated with the plasma membrane or was still freely diffusing in the cytosol. From the number of molecules that inserted during the recovery time we quantified the insertion kinetics: on average, membrane-proximal molecules within the evanescent field needed ∼400 ms to be inserted. The average lifetime of Lck in the plasma membrane was estimated at 50 s; together with the mobility of 0.26 μm2/s this provides sufficient time to explore the surface of the whole T cell before dissociation into the cytosol. Experiments on palmitoylation-deficient Lck mutants yielded similar on-rates, but substantially increased off-rates. We discuss our findings based on a model for the plasma membrane association and dissociation kinetics of Lck, which accounts for reversible palmitoylation on cysteine 3 and 5.  相似文献   
166.
The effectiveness of rapid and controlled heating of intact tissue to inactivate native enzymatic activity and prevent proteome degradation has been evaluated. Mouse brains were bisected immediately following excision, with one hemisphere being heat treated followed by snap freezing in liquid nitrogen while the other hemisphere was snap frozen immediately. Sections were cut by cryostatic microtome and analyzed by MALDI‐MS imaging and minimal label 2‐D DIGE, to monitor time‐dependent relative changes in intensities of protein and peptide signals. Analysis by MALDI‐MS imaging demonstrated that the relative intensities of markers varied across a time course (0–5 min) when the tissues were not stabilized by heat treatment. However, the same markers were seen to be stabilized when the tissues were heat treated before snap freezing. Intensity profiles for proteins indicative of both degradation and stabilization were generated when samples of treated and nontreated tissues were analyzed by 2‐D DIGE, with protein extracted before and after a 10‐min warming of samples. Thus, heat treatment of tissues at the time of excision is shown to prevent subsequent uncontrolled degradation of tissues at the proteomic level before any quantitative analysis, and to be compatible with downstream proteomic analysis.  相似文献   
167.
目的:应用对比剂动力学时间分辨成像(Time Resolved Imaging of Contrast Kinetics,TRICKS)技术增强磁共振血管成像(MRangiography,MRA)及弥散加权成像(Diffusion Weighted Imaging,DWI)技术活体动态监测兔VX2肌肉肿瘤生物学生长与血管生成,探讨肿瘤血管生成与肿瘤生长的关系。方法:30只新西兰白兔,每只均在右后腿肌肉内接种VX2肿瘤细胞1×107建立肿瘤模型。分别在肿瘤接种后第4、7、10、13、16天(每个时间点6只)分别进行T1WI、T2WI、DWI、TRICKS动态增强MRA及T1WI增强延迟扫描,活体监测兔VX2肌肉肿瘤血管生成,肿瘤标本HE及CD31免疫组化染色进行验证。两位医师双盲法分别测量不同生长点肿瘤的长、短径及体积,并与大体病理标本比较;测定TRICKS增强动态MRA所能显示肿瘤血管的最小直径及形态变化;观察ADC值变化与肿瘤生长的关系。结果(:1)ADC值随着肿瘤体积的长大而逐渐增大。(2)MRI活体测定肿瘤大小与病理大体标本所测算肿瘤体积的差异无显著性。(3)TRICKS增强MRA动态显示肿瘤血管的最小...  相似文献   
168.
目的:探讨脑囊虫不同分期、分型的磁共振表现及应用价值。方法:收集脑囊虫病例127例,行MR轴、矢、冠位扫描及增强扫描。结果:脑囊虫分型:单发小囊型44例,单发大囊型6例,多发小囊型34例,脑实质钙化型18例;脑室型11例;蛛网膜下腔型7例;混合型7例。脑囊虫分期:活动期32例,退变死亡期49例,钙化期18例,混合期28例。结论:磁共振扫描能够清晰显示各种细节,是诊断脑囊虫的有力手段。  相似文献   
169.
双指数模型在高b值弥散加权成像中的初步研究   总被引:1,自引:0,他引:1  
目的采用双指数分析模型探讨弥散加权信号强度的衰减规律,揭示脑组织的弥散信息。材料和方法对豆状核、内囊、额叶自质、丘脑等感兴趣区的每一像素,使用Matlab优化工具箱中的lsqcurvefit()函数对b值从500s/mm。到3500s/mm。共计7个b值图像的信号强度值进行拟合,并与单指数拟合的结果进行比较。结果双指数模型对信号强度的拟合优于单指数模型,并能获得三个新参数。结论双指数模型能更好的拟合高b值时图像信号强度.所得的三个参数能从不同角度提供大脑的弥散信息.但其生理基础有待于进一步研究。  相似文献   
170.
PurposeTo explore the role of Diffusion Tensor Imaging in preoperative glioma grading, as well as in differentiation between gliomas and metastatic brain tumors. We measured diffusion tensor variables in enhancement and edema regions, which were compared between the different subject groups.Materials and methodsWe performed DTI in 48 patients (11 Low Grade Gliomas, 27 High Grade Gliomas, 10 Single Metastatic brain tumors). We measured FA, λ1, λ2, λ3, ADC, Cl, Cp, Cs, RA, and VR in enhancing portions of tumors and edema regions. Additionally, ratios of enhancement to edema values were created for each variable.ResultsIn peritumoral edema, Cl and RA were proven to be significantly different in pair-wise comparisons, in addition to ADC, Cp, Cs and VR in enhancement regions. Enhancement to edema values were significantly different as well.ConclusionDiffusion tensor indices could be used for the differentiation between low and high grade gliomas, as well as for distinction between gliomas and metastases.  相似文献   
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