蝙蝠葛酚性碱对BxPC-3裸鼠移植瘤Ptch1、Smo蛋白表达的影响

目的:通过观察蝙蝠葛酚性碱(Phenolic alkaloids from Menisphermum dauricum PAMD)对胰腺癌细胞株Bx PC-3裸鼠移植瘤的抑制情况,及其对裸鼠移植瘤Hedgehog信号通路中关键分子膜受体Patched 1(Ptch1)、偶联受体Smothened(Smo)基因、蛋白表达的影响,探讨其作用机制。方法:将30只裸鼠随机选择6只作为空白对照组,其余24只裸鼠接种人源性胰腺癌细胞株Bx PC-3细胞24小时后,随机分为4组:模型组、5-氟尿嘧啶组(5-FU)、PAMD高、低剂量组,每组6只。连续给药3周后,取出肿瘤组织进行抑瘤率计算,行免疫组化(IHC)、实时定量PCR和Western blot三种方法检测裸鼠移植瘤组织中Ptch1、Smo基因及蛋白的表达影响。结果:①抑瘤率:PAMD低、高剂量组和5-FU组与模型组比较,对胰腺癌裸鼠移植瘤均有不同程度的抑制作用,抑瘤率分别为36.14%、55.88%和30.88%,具有统计学意义(P0.05)。其中以PAMD高剂量组治疗效果最好,差异具有极显著统计学意义(P0.01)。②免疫组化:模型组与各治疗组比较,Ptch1和Smo蛋白均呈现高表达,而各治疗组Ptch1蛋白和Smo蛋白表达均出现不同程度的下调,差异具有统计学意义(P0.05);同组Ptch1和Smo蛋白相互之间具有相同的表达趋势,其中PAMD高剂量组Ptch1和Smo蛋白表达下调最明显,差异有极显著的统计学意义(P0.01)。③实时定量PCR:PAMD低、高剂量组与模型组比较,Ptch1蛋白相对表达量均有不同程度的降低,差异具有极显著统计学意义(P0.01);而Smo蛋白表达量降低但不明显,差异具有统计学意义(P0.05),其中以PAMD高剂量组表达效果最明显,差异具有极显著的统计学意义(P0.01)。④Western blot结果与上述趋势相一致。结论:PAMD可通过降低Hedgehog信号通路中Ptch1、Smo关键蛋白的含量,诱导肿瘤细胞发生凋亡,从而起到抑制BxPC-3裸鼠移植瘤的生长,延缓胰腺癌的发生发展。     

Prolonged Alzheimer-like tau hyperphosphorylation induced by simultaneous inhibition of phosphoinositol-3 kinase and protein kinase C in N2a cells

Co-injection of wortmannin (inhibitor of phosphatidylinositol-3 kinase, PI3K) and GF109203X(inhibitor of protein kinase C, PKC) into the rat brain was found to induce spatial memory deficiency and enhance tau hyperphosphorylation in the hippocampus of rat brain. To establish a cell model with durative Alzheimer-like tau hyperphosphorylation in this study, we treated N2a neuroblastoma cells with wortmannin and GF109203X separately and simultaneously, and measured the glycogen synthase kinase 3 (GSK-3)activity by y-32p-labeling and the level of tau phosphorylation by Western blotting. It was found that the application of wortmannin alone only transitorily increased the activity of GSK-3 (about 1 h) and the level of tau hyperphosphorylation at Ser^396/Ser^404 and Ser^199/Ser^202 sites (no longer than 3 h); however, a prolonged and intense activation of GSK-3 (over 12 h) and enhanced tau hyperphosphorylation (about 24 h) were observed when these two selective kinase inhibitors were applied together. We conclude that the simultaneous inhibition of PI3K and PKC can induce GSK-3 overactivation, and further strengthen and prolong the Alzheimerlike tau hyperphosphorylation in N2a cells, suggesting the establishment of a cell model with early pathological events of Alzheimer‘s disease.     

糖尿病大鼠脑GSK-3与PP-2A失调诱导tau蛋白过度磷酸化

探讨胰岛素缺乏的糖尿病大鼠皮层糖原合酶激酶-3(GSK-3)及蛋白磷酯酶-2A(PP-2A)变化及其对tau蛋白磷酸化的作用.用链脲佐菌素(streptozotocin,STZ)建立胰岛素缺乏的糖尿病大鼠模型,用放射性配体结合实验检测了GSK-3和PP-2A的活性,蛋白质印迹检测了tau蛋白的磷酸化水平及PP-2A的表达.结果提示:在糖尿病大鼠皮层,GSK-3活性升高,PP-2A活性及表达降低,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化.应用GSK-3的选择性抑制剂Li2CO3后,GSK-3活性降低,PP-2A活性及表达恢复,tau蛋白在Ser198/Ser199/Ser202和Ser396/Ser404位点磷酸化水平降低.研究提示:糖尿病大鼠皮层GSK-3升高可能抑制PP-2A的活性,升高的GSK-3和降低的PP-2A协同促进tau蛋白的磷酸化.     

Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cells

We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (> 100 microM) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 microM) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine oxidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents.     

槲皮素诱导Hep G2自噬与胞内游离Ca2+浓度的变化相关

槲皮素具有诱导细胞自噬、抑制肿瘤细胞增殖等抗癌功能,但其诱导细胞自噬的分子机制还不太清楚. 本文通过激光共聚焦显微镜观察槲皮素对Hep G2细胞自噬的影响; Fluo-3 AM和Cyto-IDTM Green Detection Reagent染色标记, 流式细胞术测定了槲皮素对Hep G2细胞内游离钙离子浓度［Ca2+］_i 及Ca2＋螯合剂BAPTA-AM对自噬水平的影响. 探讨了槲皮素诱导人肝癌细胞 Hep G2自噬过程中［Ca2+］i的变化. 结果表明, 在槲皮素较低浓度范围内(0 ～ 50 μg/mL), 可明显抑制Hep G2细胞增殖, 并以剂量依赖方式诱导细胞自噬. 同时发现,槲皮素刺激Hep G2细胞可使［Ca2+］i明显增加, 进而促进自噬. 而当胞内Ca2＋螯合剂 BAPTA-AM存在时, 细胞的自噬水平受到一定的抑制. 这些结果表明,细胞内［Ca2+］i的升高可促进自噬, ［Ca2+］_i 的降低可能会抑制自噬. Hep G2细胞自噬与细胞内游离钙离子浓度的变化有关系.     

PKC and Raf-1 inhibition-related apoptotic signalling in N2a cells

In this study, a neuroblastoma N2a cell line was applied to investigate mechanisms of apoptosis induced either by selective inhibition of protein kinase C (PKC) by low amounts of staurosporine (STS(10) ) or by inhibition PI3-K after wortmannin (WM) treatment. We present evidence that, in the absence of serum in the medium, decreased phosphorylation of Raf-1 and BAD112, as well as Akt and BAD136, proteins and their translocation to mitochondria coincided with STS10 - or WM-induced apoptosis, respectively. Concomitantly, release of cytochrome c into the cytosol indicated a BCL-2-dependent mode of cell death after both treatments. Furthermore, in typical 'gain of function' experiments, cells with overexpression of permanently active Raf-1 or Akt transgenes displayed a significantly higher and independent resistance to either STS10 or WM. Thus, our results indicate that PKC/Raf-1/BAD112, as well as PI3-K/Akt/BAD136 signalling pathways, are both necessary for N2a cell survival and thus are unable to functionally substitute for each other as long as the cells do not receive additional signal(s) derived from serum. However, in the presence of serum, undefined trophic signal(s) can stimulate cross-talk between these two pathways at a level upstream from Raf-1 and Akt phosphorylation. In this case, only simultaneous inhibition of PKC and PI3-K is able to induce apoptosis.     

The Contribution of TRPV4-Mediated Calcium Signaling to Calcium Homeostasis in Endothelial Cells

A large variety of cation transport systems are involved in the regulation of calcium homeostasis in endothelial cells. The focus of the present study is to determine the contribution of nonselective cation channels from the TRP (transient receptor potential) family to cellular calcium homeostasis of porcine aortic endothelial cells (PAEC). One member of the TRPV (vanniloid) subfamily, TRPV4, has previously been shown to be involved in cation transport induced by a large variety of stimulations including osmolarity, temperature, mechanical stress, and phosphorylation. Here, we demonstrate the existence of several TRP proteins, including TRPV4, in PAEC using RT-PCR. To test whether this channel is functional, we performed FURA-2 calcium measurements and whole-cell patch-clamp experiments. We observed the induction of large calcium signals following mechanical stress, altered extracellular temperature, and the selective TRPV4 activator 4-α -PDD. These effects were diminished in the presence of the TRPV4 inhibitor miconazole, suggesting the involvement of this channel in mediating endothelial calcium signals. The large amounts of transported calcium and the short signaling ways suggest a potentially important role of this channel in many physiological processes.     

Phosphorylation of tau at serine 416 by Ca2+/calmodulin-dependent protein kinase II in neuronal soma in brain

It is well known that tau is a good in vitro substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). However, it is not clear at present whether CaM kinase II phosphorylates tau in vivo or not. Serine 416, numbered according to the longest human tau isoform, has been reported to be one of the major phosphorylation sites by CaM kinase II in vitro. In this study, we produced a specific antibody against tau phosphorylated at serine 416 (PS416-tau). Immunoblot analysis revealed that the antibody reacted with tau in the rat brain extract which was prepared in the presence of protein phosphatase inhibitors. Developmental study indicated that serine 416 was strongly phosphorylated at early developmental stages in rat brain. We examined the localization of PS416-tau in primary cultured hippocampal neurons and the immortalized GnRH neurons (GT1-7 cells), which were stably transfected with CaM kinase IIalpha cDNA. Immunostaining of these cells indicated that tau was phosphorylated mainly in neuronal soma. Interestingly, tau in neuronal soma in Alzheimer's disease (AD) brain was strongly immunostained by the antibody. These results suggest that CaM kinase II is involved in the accumulation of tau in neuronal soma in AD brain.     

激光扫描共聚焦显微镜观察胰蛋白酶对肺上皮细胞游离钙离子的影响

目的：研究PAR-2激动剂SLIGKV和tc-LIGRLO、胰蛋白酶及其抑制剂对H292肺上皮细胞[Ca^2＋]i的影响.方法：应用Fluo-3/AM 荧光标记技术和激光扫描共聚焦显微镜（LSCM） 检测不同因素处理的H292肺上皮细胞[Ca^2＋]i.结果：胰蛋白酶、SLIGKV、tc-LIGRLO均能引发H292细胞[Ca^2＋]i的增加,平均荧光强度分别比加入药物前增加267%,60%和37%.胰蛋白酶抑制剂大豆胰蛋白酶抑制剂（SBTI）和α1-抗胰蛋白酶（α1-AT）可以抑制胰蛋白酶诱导的细胞[Ca^2＋]i的增加.结论：PAR-2可以介导H292肺上皮细胞[Ca^2＋]i的释放增加,胰蛋白酶抑制剂可以抑制胰蛋白酶诱导的细胞[Ca^2＋]i的增加.