The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, we found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons, as the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics.     

A bromine-catalysis-synthesized poly(3,4-ethylenedioxythiophene)/graphitic carbon nitride electrochemical sensor for heavy metal ion determination

In this paper, poly(3,4-ethylenedioxythiophene)/graphitic carbon nitride (PEDOT/g-C₃N₄) composites were prepared by the bromine catalysed polymerization (BCP) method with varying weight ratios of monomer to g-C₃N₄. For comparison, solid-state polymerization (SSP) and metal oxidative polymerization (MOP) methods were also used for the synthesis of PEDOT/g-C₃N₄ composites. Electrochemical determination of heavy metal ions (Cd²⁺ and Pb²⁺) was carried out by differential pulse voltammetry (DPV) on composite-modified glass carbon electrodes (GCEs), which were prepared by different methods. The obtained composites were analysed by Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible absorption spectroscopy (UV-vis), X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The results showed that the bromine catalysed polymerization (BCP) method is an effective way to prepare the PEDOT/g-C₃N₄ composite, and the combination of PEDOT with g-C₃N₄ can improve the electrochemical activity of electrode materials. And, the composite from the BCP method modified electrode (PEDOT/10 wt% g-C₃N₄/GCE) exhibited the widest linear responses for Cd²⁺ and Pb²⁺, ranging from 0.06–12 μM and 0.04–11.6 μM with detection limits (S/N = 3) of 0.0014 μM and 0.00421 μM, respectively.

The PEDOT/g-C₃N₄ composite prepared by a Br₂-catalyzed polymerization method exhibited the widest linear electrochemical responses for Cd²⁺ and Pb²⁺.     

囊型包虫病患者外周血单核细胞诱导的树突状细胞形态及表型特点

目的研究囊型包虫病患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)体外诱导的树突状细胞(dendritic cell,DC)的形态及其细胞表型特点。方法囊型包虫病患者组(cystic echinococcosis,CE)15例,健康志愿者对照组(healthy donor,HD)15例,采外周血(肝素抗凝)分离单个核细胞后贴壁培养,施加重组细胞因子培养6d,诱导成DC,采用倒置显微镜和扫描电镜观察细胞形态,流式细胞术检测CD86、HLA-DR、CD1a、CD80的表达情况。结果与HD组比较,CE组PBMC诱导的DC表面的树突状突起较少,且细胞表面的CD86、HLA-DR、CD1a、CD80表达降低(P0.05)。结论CE组患者PBMC诱导的DC形态不典型,表面分子的表达降低,细胞活力降低,对T细胞的刺激能力减弱。     

细粒棘球蚴囊液对体外培养小鼠脾细胞Foxp3及Smad4基因表达的影响

目的观察细粒棘球蚴囊液对体外培养BABL/c小鼠脾脏细胞中调节性T细胞(Treg细胞)相对特异分子Foxp3(叉头蛋白3)及TGF-β1(转化生长因子-β1)下游信号通路Smad4表达的影响。方法以BABL/c小鼠作为脾细胞供者,用研磨法分离脾脏细胞,随机分为实验组(与细粒棘球蚴囊液共同培养)和对照组,取1×105个细胞接种于96孔板上,分别在1、3、6和12 h提取RNA,实时荧光定量PCR(qRT-PCR)检测Foxp3以及Smad4的基因表达。结果 qRT-PCR显示,细粒棘球蚴囊液处理1、3和12 h,实验组Foxp3表达量分别为4.577±0.317、8.517±0.978和7.406±0.822,对照组分别为9.274±0.451、3.297±0.408和2.464±0.328,差异均有统计学意义(P<0.05);细粒棘球蚴囊液处理1 h和12 h,实验组Smad4表达量分别为3.862±1.417和1.690±0.248,对照组分别为1.689±0.221和3.600±1.081,差异有统计学意义(P<0.05),且Foxp3与Smad4二者之间存在负相关(r=-0.991,P<0.05)。结论...     

Genistein对人鼻咽癌细胞系CNE抑制增殖和促进凋亡的研究

鼻咽癌是头颈肿瘤中具有鲜明流行病学、组织病理学、临床和治疗学特征的一种肿瘤。研究表明癌细胞的增殖活性是影响鼻咽癌生长进展的主导因素，肿瘤细胞的增殖活性与肿瘤的生物学行为与预后密切相关。Genistein（4，5，7-三羟基异黄酮，又名金雀异黄素）是一种广谱的蛋白酪氨酸激酶（protein tyrosine kinase，PTK）抑制剂。[第一段]     

人外周血树突状细胞体外稳定培养方法的建立及与磁珠法的比较

目的 建立一种稳定的人外周血树突状细胞(DCs)体外培养的方法,并与磁珠分选法进行比较.方法 通过密度梯度离心法分离出志愿者的外周血单个核细胞(PBMC),再分别应用磁珠分选法、贴壁法对PBMC进行培养,应用重组人集落刺激因子(rhGM-CSF)、重组人白细胞介素-4(rhIL-4)诱导获得DCs.倒置显微镜观察细胞形态变化,并分别在第3、5、6天用台盼蓝染色法进行细胞活力检测;经过1、2、5 h的贴壁培养后,应用流式细胞仪检测单核细胞表面CD14、CD1a、HLA-DR的表达以确定最佳贴壁时间;经人重组细胞因子诱导培养后,对所获得的细胞检测CD14、CD1a、CD86、CD83、HLA-DR的表达.采用同种混合淋巴细胞反应,评价DCs刺激T淋巴细胞增殖的能力.结果 经贴壁2 h后诱导培养的DCs形态较典型.磁珠分选法获得的DCs第5、6天细胞活力[(53.333±5.774)%、(38.333±7.638)%]明显低于第3天[(68.667±3.215)%,P均＜0.05];贴壁培养法获得的DCs第3、5、6天的细胞活力[(92.667±3.055)%、(94.000±1.000)%和(94.667±1.528)%]比较,差异无统计学意义(F=0.737,P＞0.05);贴壁培养法获得的DCs第3、5、6天细胞活力均高于磁珠分选法(t值分别为9.374、12.021、12.527,P均＜0.05).PBMC经磁珠分选前后CD14的阳性表达率分别为(32.457±12.351)%、(41.914±14.858)%,二者比较差异无统计学意义(t=1.295,P＞0.05).单核细胞表面CD14的阳性表达率在培养2 h时[(35.267±4.658)%]高于培养1、5 h时[(15.033±6.189)%、(21.233±4.895)%,P均＜0.05].培养第6天,DCs表面CD14的阳性表达率[(2.200±1.356)%]较第1天[(32.328±14.517)%]明显下降(t=5.467,P＜0.05),CD1a的阳性表达率[(43.371±16.250)%]较第1天[(12.300±6.223)%]显著升高(t=2.545,P＜0.05);而CD86、CD83、HLA-DR的阳性表达率[(16.857±5.686)%、(9.343±5.230)%、(72.800±17.881)%]与第1天[(12.550±16.758)%、(6.250±1.323)%、(64.671±15.588)%]比较,差异无统计学意义(t值分别为0.652、1.137、0.907,P均＞0.05).同种混合淋巴细胞反应,随着淋巴细胞的增多,增殖能力下降.磁珠分选法中,DCs与淋巴细胞的比例为1:50、1:100时,细胞增殖能力(1.502±0.055、1.507±0.029)较1:10时(1.859±0.049)降低(P均＜0.05);贴壁培养法中,DCs与淋巴细胞的比例为1:100时,细胞增殖能力(1.545±0.066)较1:10时(2.015±0.301)降低(P＜0.05).在DCs与淋巴细胞的比例相同时,两种方法得到的DCs在刺激T淋巴细胞方面的能力相近(P＞0.05).结论 与磁珠分选法比较,贴壁培养2 h后的人外周血PBMC再行诱导可获得形态与功能较优的DCs,且此法稳定、简便、经济,是一种适于基础、临床研究的DCs体外培养方法.     

A natural scavenger of peroxynitrites, rosmarinic acid, protects against impairment of memory induced by Abeta(25-35)

Peroxynitrite (ONOO⁻)-mediated damage is regarded to be responsible for the cognitive dysfunction induced by amyloid beta protein (Aβ) in Alzheimer's disease (AD). In the present study, we examined the protective effects of rosmarinic acid (RA), a natural scavenger of ONOO⁻, on the memory impairment in a mouse model induced by acute i.c.v. injection of Aβ_25–35. Mice daily received i.p. several doses of RA after the injection of Aβ_25–35. RA prevented the memory impairments induced by Aβ_25–35 in the Y maze test and novel object recognition task. RA, at the effective lowest dose (0.25 mg/kg), prevented Aβ_25–35-induced nitration of proteins, an indirect indicator of ONOO⁻ damage, in the hippocampus. At this dose, RA also prevented nitration of proteins and impairment of recognition memory induced by ONOO⁻-i.c.v.-injection. Co-injection of the non-memory-impairing dose of ONOO⁻ with Aβ_25–35 blocked the protective effects of RA (0.25 mg/kg). These results demonstrated that the memory protective effects of RA in the neurotoxicity of Aβ_25–35 is due to its scavenging of ONOO⁻, and that daily consumption of RA may protect against memory impairments observed in AD.