Acylphosphatase overexpression triggers SH-SY5Y differentiation towards neuronal phenotype

An acylphosphatase (AcPase) overexpression study was carried out on SH-SY5Y neuroblastoma cells, using a green fluorescent fusion protein (AcP-GFP), with GFP acting as a reporter protein. The cellular proliferation rate was significantly reduced by overexpression of AcPase by a factor of ten. In contrast, clones transfected with two inactive AcPase mutants showed a growth rate comparable to control cells. This suggests that AcPase catalyzes the proliferative down-regulation. AcPase-overexpressing clones showed a physiological mortality rate as assessed by an MTT reduction test and by evaluation of necrotic markers. DNA fragmentation analysis and assays of caspase-3 and poly (ADP-ribose) polymerase (PARP)-active fragments showed no evidence of any apoptotic pattern. AcPase overexpression led to a marked increase in PARP activity as well as Bcl-2 content; these are commonly up-regulated during differentiative processes in neuronal cells. In fact, the typical differentiation marker, growth-associated-protein 43, was significantly up-regulated. Microscopic observations also showed a clear increase in the differentiative phenotype in AcPase-overexpressing cells. Our results clearly show that AcPase plays a primary causative role in neuronal differentiation.Received 3 May 2004; accepted 25 May 2004     

Membrane resting potentials in cultured mouse neuroblastoma cells

Summary Membrane resting potentials (MRP) were measured systematically in cultured mouse N₂A neuroblastoma cells: 1) in the logarithmic growth phase; 2) in subconfluent cultures; 3) in confluent cultures; 4) after dBcAMP had induced morphological differentiation. Neurite extension was accompanied by a significant increase in MRP as compared to the appropriate controls. No significant differences in MRP were observed with regard to the different growth phases.     

Neuronal growth inhibitory factor inhibits pheochromo-cytoma PC12 in vitro

Neuronal growth inhibitory factor (GIF),named Metaliothioneins-Ⅲ (MT-Ⅲ), is the first protein validated to be capable of inhibiting the growth of neurons in nervous system. We have detected the effects of recombinant GIF on the growth of neuroblastoma SY5Y and pheochromocytoma PC12 by the MTT (Thiazolyl blue) reduction assay. Recombinant GIF inhibited PC12 in vitro; the inhibitory rate was about 25% when GIF was at 100 mg/L; and the inhibitory rate was about 50% when GIF was at 300 mg/L. It is shown that PC12 could serve as a proper model for detecting neuronal growth inhibitory activity of GIF. Recombinant GIF did not inhibit neuroblastoma SY5Y in vitro, a common model of neuroma; it is also shown that GIF could not inhibit neuromata extensively. The reason for GIF inhibiting PC12 may be that PC12 have some properties of cholinergic neuron. It must play an important role in discovering the mechanism of GIF's neuronal growth inhibitory activity.``     

绿色荧光蛋白标记的NTE活性域表达载体的构建及表达

利用含有特定限制性内切酶识别位点的引物,通过聚合酶链式反应扩增出人神经病靶标酯酶活性域的编码序列,经T载体克隆测序正确后,双酶切回收特异片段定向插入到增强型绿色荧光表达载体pEGFP-N3中,通过酶切反应鉴定,构建了绿色荧光蛋白标记的神经病靶标酯酶活性域的融合表达载体pNEST-EGFP.采用脂质体转染的方法将其转染到人神经瘤母瘤细胞SH-SY5Y中,用荧光显微镜观察发现神经病靶标酯酶活性域分布于细胞质中,而且没有导致内质网膜的聚集,表明表达载体成功构建和表达.     

Prolonged copper depletion induces expression of antioxidants and triggers apoptosis in SH-SY5Y neuroblastoma cells

SH-SY5Y neuroblastoma cells were cultured for up to three serial passages in the presence of the copper chelator triethylene tetramine (Trien). The copper-depleted neuroblastoma cell line obtained showed decreased activities of the copper enzymes Cu, Zn superoxide dismutase and cytochrome c oxidase with concomitant increases in reactive oxygen species. Mitochondrial antioxidants (Mn superoxide dismutase and Bcl-2) were up-regulated. Overexpression and activation of p53 were early responses, leading to an increase in p21. Eventually, copper-depleted cells detached from the monolayer and underwent apoptosis. Activation of up-stream caspase-9, but not caspase-8, suggested that apoptosis proceeds via a mitochondrial pathway, followed by caspase-3 activation. The addition of copper sulfate to the copper-depleted cells restored copper enzymes, normalized antioxidant levels and improved cell viability. We conclude that prolonged copper starvation in these replicating cells leads to mitochondrial damage and oxidative stress and ultimately, apoptosis.Received 24 April 2003; accepted 23 May 2003     

Selectivity of action between pyrethroids and combined DDT-pyrethroid insecticides on Na+ influx into mammalian neuroblastoma

Summary Several of the most active synthetic pyrethroid insecticides in the presence of sea anemone toxin II, induced a dose related influx of sodium ion into the C9 mouse neuroblastoma. The influx of sodium ion into this mammalian cell did not take place with a DDT analogue, EDO and several new combined DDT-pyrethroid insecticides, although these have been reported to cause excess sodium influx into arthropod axons, related to their insecticidal activity. This difference between species in the action of the new insecticides at the nerve sodium channel explains their low mammalian toxicity.     

Gradient distribution pattern of muscarinic receptors in N1E 115 mouse neuroblastoma cells

Summary The distribution pattern of muscarinic receptors in N1E 115 mouse neuroblastoma cells after linear and non-linear gradient centrifugation was investigated. In untreated cells, at least two forms of the receptors, with different densities, were found.Bursary of the IWONL.     

两种帕金森体外模型的构建及相关研究

为检测a-突触核蛋白(a-Synuclein)在人神经元母细胞瘤细胞(SH-SY5Y)中的表达,构建人野生型(WT)和A53T突变型a-Synuclein质粒(p EGFP-SNCA-WT和p EGFP-SNCA-A53T),经酶切鉴定无误后转染SH-SY5Y细胞,并对转染完成的细胞进行嘌呤霉素筛选,然后通过PCR法扩增转染后SH-SY5Y细胞的a-Synuclein片段,并对其进行测序,检测目的基因,运用Western blot法检测转染后SH-SY5Y细胞中a-Synuclein的表达.酶切鉴定结果表明p EGFP-SNCA-WT和p EGFP-SNCA-A53T质粒构建成功,嘌呤霉素筛选和转基因细胞a-Synuclein片段测序结果显示慢病毒表达载体能成功的整合到SH-SY5Y细胞基因组中,Western blot结果表明,转染后的SH-SY5Y细胞能成功的过表达a-Synuclein.a-Synuclein慢病毒表达载体构建成功,并且SH-SY5Y细胞作为宿主细胞能够表达a-Synuclein.为今后帕金森病(PD)体外模型的建立及帕金森病发病机制的研究奠定了基础.     

Accessibility of a critical prion protein region involved in strain recognition and its implications for the early detection of prions

Human prion diseases are characterized by the accumulation in the brain of proteinase K (PK)-resistant prion protein designated PrP27 – 30 detectable by the 3F4 antibody against human PrP109 – 112. We recently identified a new PK-resistant PrP species, designated PrP^*20, in uninfected human and animal brains. It was preferentially detected with the 1E4 antibody against human PrP 97 – 108 but not with the anti-PrP 3F4 antibody, although the 3F4 epitope is adjacent to the 1E4 epitope in the PrP^*20 molecule. The present study reveals that removal of the N-terminal amino acids up to residue 91 significantly increases accessibility of the 1E4 antibody to PrP of brains and cultured cells. In contrast to cells expressing wild-type PrP, cells expressing pathogenic mutant PrP accumulate not only PrP^*20 but also a small amount of 3F4-detected PK-resistant PrP27 – 30. Remarkably, during the course of human prion disease, a transition from an increase in 1E4-detected PrP^*20 to the occurrence of the 3F4-detected PrP27 – 30 was observed. Our study suggests that an increase in the level of PrP^*20 characterizes the early stages of prion diseases. Received 17 October 2007; received after revision 5 December 2007; accepted 14 December 2007