钙通道和Na+/H+交换在表皮生长因子促肝癌细胞生长中的作用

目的许多生长因子如表皮生长因子(EGF),与肿瘤的发生密切相关.EGF与表皮生长因子受体(EGFR)结合,通过一系列的信息传导,导致肝癌细胞的增生.但受体后的信息传导机制尚不清楚.本实验探讨酪氨酸激酶、蛋白激酶C、Na+/H+交换、钙调蛋白和电压依赖性钙通道在EGF促肝癌细胞生长中的作用.方法本研究于无血清RPMI1640中培养肝癌细胞SMMC7721,采用3H-Thymidine(3H-TdR)掺入的方法,检测肝癌细胞DNA合成速率,研究酪氨酸激酶、蛋白激酶C、Na+/H+交换、钙调蛋白和电压依赖性钙通道在EGF促肝癌细脆生长中的作用.结果EGF 10-9M对肝癌细脆的生长有极显著促进作用,与对照组比较差异有显著意义(P＜0.05),酪氮酸激酶阻滞剂Genistein对EGF的促肝癌细胞生长作用具有极显著抑制作用(P＜0.001).钙调蛋白阻滞剂W-7、蛋白激酶C阻滞剂H-7和Na+/H+交换阻滞剂amiloride对EGF的促肝癌细胞生长作用具有显著抑制作用(P＜0.001,P＜0.01,P＜0.05),而对基础状态细胞的3H-TdR掺入值无显著影响(P＞0.05).电压依赖性钙通道阻滞剂Varapamil对BGF的促肝癌细胞生长作用无显著抑制作用(P＞0.05),对基础状态细胞的3H-TdR掺入值亦无显著影响(P＞0.05).结论结果显示,酪氨酸激酶、蛋白激酶C、Na+/H+交换及依赖钙-钙调蛋白途径在BGF的作用中起关键作用.电压依赖性钙通道与EGF的作用无关.     

二甲双胍调节糖尿病大鼠心房小电导钙激活钾通道亚型蛋白表达的信号通路

目的探讨蛋白激酶A(PKA)/钙调蛋白Ⅱ(CaMKⅡ)信号通路在二甲双胍(Met)调节2型糖尿病(T2DM)大鼠心房小电导钙激活钾通道亚型KCa2.2和KCa2.3蛋白表达中的作用。方法健康雄性Wistar大鼠40只,随机选8只喂普通饲料为对照组(Con组),另32只喂高脂高糖饲料联合腹腔注射小剂量链脲佐菌素构建T2DM大鼠模型后,再随机分为DM组、Met组、H-89组(腹腔注射PKA抑制剂H-89)和KN-93组(腹腔注射CaMKⅡ抑制剂KN-93),每组8只。用ELISA检测大鼠心房组织PKA活性,qRT-PCR检测CaMKⅡmRNA表达,Western blot和免疫组织化学检测KCa2.2、KCa2.3和磷酸化CaMKⅡ(p-CaMKⅡ)蛋白表达。结果 Con组、DM组、Met组和H-89组PKA活性分别为0.74±0.04、0.50±0.05、0.69±0.03和0.48±0.03。与DM组比较,Met组PKA活性明显提升(P<0.01);与Met组比较,H-89组显著抑制PKA活性(P<0.01)。Con组、DM组及Met组CaMKⅡmRNA分别为1.00±0.07、0.61±0.03和0.92±0.09。与Con组比较,DM组CaMKⅡmRNA表达明显降低(P<0.01);与DM组比较,Met组CaMKⅡmRNA表达明显增加(P<0.01)。与Con组比较,DM组心房组织p-CaMKⅡ和KCa2.2蛋白表达均明显降低,KCa2.3蛋白表达明显升高(P<0.01)。与DM组比较,Met组明显提升p-CaMKⅡ和KCa2.2蛋白表达,明显抑制KCa2.3蛋白表达(P<0.01)。与Met组比较,KN-93组和H-89组分别显著抑制p-CaMKⅡ蛋白表达和PKA活性,均显著下调KCa2.2蛋白表达,上调KCa2.3蛋白表达(P<0.01)。免疫组织化学染色显示,与Met组比较,KN-93组和H-89组均显著下调KCa2.2蛋白表达,上调KCa2.3蛋白表达(P<0.05,P<0.01),与Western blot检测结果一致。结论 Met通过激活PKA/CaMKⅡ信号通路部分修复T2DM大鼠心房KCa2.2蛋白下调和KCa2.3蛋白上调。     

The occludin and ZO-1 complex, defined by small angle X-ray scattering and NMR, has implications for modulating tight junction permeability

Tight junctions (TJs) are dynamic cellular structures that are critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, and fluids. Phosphorylation-dependent binding of the transmembrane protein occludin to the structural organizing protein ZO-1 contributes to the regulation of barrier properties; however, the details of their interaction are controversial. Using small angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, and site-directed mutagenesis experiments. we define the interface between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil (CC) domains. The interface is comprised of basic residues in PSG and an acidic region in CC. Complex formation is blocked by a peptide (REESEEYM) that corresponds to CC residues 468-475 and includes a previously uncharacterized phosphosite, with the phosphorylated version having a larger effect. Furthermore, mutation of E470 and E472 reduces cell border localization of occludin. Together, these results localize the interaction to an acidic region in CC and a predominantly basic helix V within the ZO-1 GuK domain. This model has important implications for the phosphorylation-dependent regulation of the occludin:ZO-1 complex.     

Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine‐binding site of N‐methyl‐D‐aspartate receptor

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N‐methyl‐D ‐aspartate receptor (NMDAR)‐dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR‐dependent long‐term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10–100 nM significantly enhanced LTP in a bell‐shaped dose‐response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7‐chloro‐kynurenic acid (7‐ClKN), an antagonist for glycine‐binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α‐amino‐3‐hydroxy‐5‐methylisozazole‐4‐propionate receptor (AMPAR) through activation of calcium/calmodulin‐dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1–1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose‐dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser‐657) and Src family (Tyr‐416) activities with the same bell‐shaped dose‐response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser‐657) and Src (Tyr‐416) induced by sunifiram was inhibited by 7‐ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine‐binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation. © 2013 Wiley Periodicals, Inc.     

两种肌萎缩侧索硬化基因表达谱差异性比较

 目的 比较肌萎缩侧索硬化的两种不同基因突变型(C9orf72与CHMP2B)表达谱差异来探讨该类疾病可能的发病机制及治疗靶点。方法 从GEO数据库中下载C9orf72基因突变肌萎缩侧索硬化数据集(GSE68605)及CHMP2B基因突变肌萎缩侧索硬化数据集(GSE19332)。使用R软件(3.5.0版本)、Cytoscape 3.6.1软件及在线工具(DAVID及STRING)进行数据分析。结果 从两个数据集中,获得了11个样本,其中8个为C9orf72基因突变,3个为CHMP2B基因。发现了13个差异表达基因,在GO及KEGG功能富集分析中发现仅有CALM1-3及RYR2富集在钙离子检测、通过钙离子释放的调节影响心肌的收缩功能等。其中钙调蛋白是引起C9orf72基因突变肌萎缩侧索硬化及CHMP2B基因突变肌萎缩侧索硬化差异的关键蛋白。结论 CALM基因在C9orf72基因突变肌萎缩侧索硬化患者中高表达,钙调蛋白可能是诊断及治疗C9orf72基因突变肌萎缩侧索硬化患者的重要靶点。     

Phosphorylation of neuronal nitric oxide synthase at Ser847 in the nucleus intermediolateralis after spinal cord injury in mice

We previously demonstrated that Ca2+/calmodulin (CaM)-dependent protein kinase IIalpha (CaM-KIIalpha) can phosphorylate neuronal nitric oxide synthase (nNOS) at Ser847 and attenuate NOS activity in neuronal cells. In the present study we focused on chronological alteration in levels and cellular location of nNOS, phosphorylated (p)-Ser847-nNOS (NP847), CaM-KII and p-Thr286-CaM-KIIalpha following spinal cord injury (SCI) in mice. Western blot analysis showed nNOS to be significantly phosphorylated at Ser847 from 3 h after SCI, peaking at 24 h and gradually decreasing thereafter, and CaM-KII to be colocalized with nNOS after SCI. Immunohistochemical analysis revealed that SCI causes an increase in both NP847 and p-Thr286-CaM-KIIalpha in the nucleus intermediolateralis. These findings suggest that SCI induces p-Thr286-CaM-KIIalpha, which phosphorylates the nNOS at Ser847 in the nucleus intermediolateralis where NO is thought to play a role as a neurotransmitter in autonomic preganglionic neurons. Thus, the NP847 signaling pathway might be involved in the autonomic failure which occurs immediately after SCI.     

Lead alters intracellular protein signaling and suppresses pro-inflammatory activation in TLR4 and IFNR-stimulated murine RAW 264.7 cells,in vitro

Lead (Pb) is a persistent environmental pollutant that has a structure and charge similar to many ions, such as calcium, that are essential for normal cellular function. Pb may compete with calcium for protein binding sites and inhibit signaling pathways within the cell affecting many organ systems including the immune system. The aim of the current study was to assess whether the calcium/calmodulin pathway is a principal target of environmentally relevant Pb during pro-inflammatory activation in a RAW 264.7 macrophage cell line. RAW 264.7 cells were cultured with 5 μM Pb(NO₃)₂, LPS, rIFNγ, or LPS+rIFNγ for 12, 24, or 48 hr. Intracellular protein signaling and multiple functional endpoints were investigated to determine Pb-mediated effects on macrophage function. Western blot analysis revealed that Pb initially modulated nuclear localization of NFκB p65 and cytoplasmic phosphorylation of CaMKIV accompanied by increased phosphorylation of STAT1β at 24 hr. Macrophage proliferation was significantly decreased at 12 hr in the presence of Pb, while nitric oxide (NO) was significantly reduced at 12 and 24 hr. Cells cultured with Pb for 12, 24, or 48 hr exhibited altered cytokine levels after specific stimuli activation. Our findings are in agreement with previous reports suggesting that macrophage pro-inflammatory responses are significantly modulated by Pb. Further, Pb-induced phosphorylation of CaMKIV (pCaMKIV), observed in the present study, may be a contributing factor in metal-induced autophagy noted in our previous study with this same cell line.     

Anterior‐posterior regionalized gene expression in the Ciona notochord

Background: In the simple ascidian chordate Ciona, the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non‐compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape, and behavior vary consistently along the anterior‐posterior (AP) axis. Results: Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non‐uniform expression in the notochord. Ezrin/radixin/moesin (ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin‐like gene (BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF‐β ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL, and TGF‐β expression are evident even before the notochord cells have intercalated into a single‐file column. Conclusions: We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression. Developmental Dynamics 243:612–620, 2014. © 2013 Wiley Periodicals, Inc.