Neuroprotective and nootropic drug noopept rescues α-synuclein amyloid cytotoxicity

Parkinson's disease is a common neurodegenerative disorder characterized by α-synuclein (α-Syn)-containing Lewy body formation and selective loss of dopaminergic neurons in the substantia nigra. We have demonstrated the modulating effect of noopept, a novel proline-containing dipeptide drug with nootropic and neuroprotective properties, on α-Syn oligomerization and fibrillation by using thioflavin T fluorescence, far-UV CD, and atomic force microscopy techniques. Noopept does not bind to a sterically specific site in the α-Syn molecule as revealed by heteronuclear two-dimensional NMR analysis, but due to hydrophobic interactions with toxic amyloid oligomers, it prompts their rapid sequestration into larger fibrillar amyloid aggregates. Consequently, this process rescues the cytotoxic effect of amyloid oligomers on neuroblastoma SH-SY5Y cells as demonstrated by using cell viability assays and fluorescent staining of apoptotic and necrotic cells and by assessing the level of intracellular oxidative stress. The mitigating effect of noopept against amyloid oligomeric cytotoxicity may offer additional benefits to the already well-established therapeutic functions of this new pharmaceutical.     

河南鼠尾草抑制体内外α-糖苷酶活性研究

本文采用96微孔板法,首次对河南鼠尾草抑制酵母和大鼠小肠α-葡萄糖苷酶活性进行研究。河南鼠尾草乙酸乙酯提取物(IC50=28.73μg/mL)和正丁醇提取物(IC50=73.90μg/mL)抑制酵母α-葡萄糖苷酶活性远高于阳性对照Acarbose(IC50=1081.27μg/mL),但只有乙酸乙酯提取物(IC50=366.79μg/mL)具有抑制大鼠小肠α-葡萄糖苷酶活性,阳性对照Acarbose未检测出其IC50。结果表明,河南鼠尾草乙酸乙酯提取物和正丁醇提取物均具有较好的酵母α-葡萄糖苷酶抑制活性,但只有乙酸乙酯提取物具有良好的大鼠小肠α-葡萄糖苷酶抑制活性。     

Apoptosis induced by (di-isopropyloxyphoryl-Trp)2-Lys-OCH3 in K562 and HeLa cells

According to the method used in our laboratory, our group synthesized (DIPP-Trp)₂-Lys-OCH₃. It inhibited the proliferation of K562 and HeLa cells in a dose-and time-dependent manner with an IC₅₀ of 15.12 and 42.23 µM, respectively. (DIPP-Trp)₂-Lys-OCH₃ induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells; the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Phosphatidylserine could significantly translocate to the surface of the membrane in (DIPP-Trp)₂-Lys-OCH₃-treated K562 and HeLa cells. The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining. It was concluded that (DIPP-Trp)₂-Lys-OCH₃ not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle. It may have arrested the K562 and HeLa cells in the G₂/M, S phases, respectively. The apoptotic pathway was pulsed at this point, resulting in the treated cells entering into programmed cell death. (DIPP-Trp)₂-Lys-OCH₃ is a potential anticancer drug that intervenes in the signalling pathway.     

Alpha-melanocyte stimulating hormone protects retinal pigment epithelium cells from oxidative stress through activation of melanocortin 1 receptor–Akt–mTOR signaling

Patients with age related macular degeneration (AMD) will develop vision loss in the center of the visual field. Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is an important contributor of AMD. In this study, we explored the pro-survival effect of α-melanocyte stimulating hormone (α-MSH) on oxidative stressed RPE cells. We found that α-MSH receptor melanocortin 1 receptor (MC1R) was functionally expressed in primary and transformed RPE cells. RPE cells were response to α-MSH stimulation. α-MSH activated Akt/mammalian target of rapamycin (mTOR) and Erk1/2 signalings in RPE cells, which were inhibited by MC1R siRNA knockdown. α-MSH protected RPE cells from hydrogen peroxide (H₂O₂)-induced apoptosis, an effect that was almost abolished when MC1R was depleted by siRNA. α-MSH-mediated S6K1 activation and pro-survival effect against H₂O₂ was inhibited by Akt inhibitors (perifosine, MK-2206 and LY294002). Further, mTOR inhibition by rapamycin, or by mTOR siRNA knockdown, diminished α-MSH’s pro-survival effect in RPE cells. Thus, Akt and its downstream mTOR signaling mediates α-MSH-induced survival in RPE cells. In summary, we have identified a new α-MSH–MC1R physiologic pathway that reduces H₂O₂-induced RPE cell damage, and might minimize the risk of developing AMD.     

地衣芽孢杆菌耐高温α-淀粉酶基因的克隆、烟草瞬时表达及转化拟南芥的研究

从地衣芽孢杆菌(Bacillus licheniformis)中克隆到耐高温α-淀粉酶基因全长, 构建了原核表达载体, 转入大肠杆菌(Escherichia coli)中, 使用IPTG于28°C诱导6小时后, 通过SDS-PAGE检测到目的蛋白, 分子量约为55 kDa, 并通过酶活力检测实验证明该蛋白具有耐高温α-淀粉酶活性。同时构建了该基因融合GFP的植物表达载体, 通过农杆菌(Agro- bacterium tumefaciens)介导瞬时转化烟草(Nicotiana tabacum)下表皮细胞并在荧光显微镜下观察, 发现在烟草下表皮细胞的细胞质和液泡中均有绿色荧光。使用I₂-KI溶液对乙醇脱色后的烟草叶片进行染色, 显色反应表明在烟草中表达的耐高温α-淀粉酶具有酶活性。最后, 采用农杆菌介导的花蕾浸泡法将重组载体转化到拟南芥(Arabidopsis thaliana)中, 筛选到稳定遗传的耐高温α-淀粉酶基因的拟南芥纯合子。研究结果为后期开展表达耐高温α-淀粉酶的转基因植物的相关研究奠定了实验基础。     

人干扰素α-2b 基因在烟草中的表达研究

应用PCR的技术从质粒pAIFN中扩增人干扰素α-2b（Human interferon α-2b,HuIFN α-2b）编码基因,将其连接到pBI121双元载体构建植物真核表达载体pBIFN;用冻融法将该载体转染根癌农杆菌LBA4404;并用叶盘浸染法转化烟草叶片,经转化的烟草叶片的组织培养,诱导愈伤获得再生植株。通过应用PCR,RT-PCR,Wes-tern blot和WISH/VSV方法检测获得的烟草再生植株,结果表明HuIFN α-2b基因已成功整合进烟草核基因组并表达出具有活性的HuIFN α-2b蛋白。本文对HuIFN α-2b基因在烟草核系统中的表达进行了研究,为进一步在烟草叶绿体系统中该基因的表达研究奠定了基础。     

茉莉酸甲酯诱导大戟三萜类代谢的研究

京大戟是多年生草本药用植物,入药部分是其干燥根,但可入药的京大戟资源由于生长缓慢以及环境污染的加剧而越发匮乏,因此解决大戟资源日益紧张的问题是当今药用植物资源开发与利用方向的重要课题。京大戟含有三萜类、二萜类、黄酮类等丰富的活性成分,一些常见药用植物的有效成分是三萜类化合物,其在抗病毒、抗肿瘤、免疫调节等方面具有很好的活性。对植物萜类物质代谢起重要作用的关键酶,如3-羟基,3-甲基戊二酰辅酶A还原酶(hmgr)、鲨烯合酶(sqs)、法尼基焦磷酸合酶(fps)的基因克隆及活性研究取得了进展和突破,但通过调控萜类物质代谢途径中关键酶基因的表达来诱导终产物合成的研究鲜有报道。通过研究大戟萜类物质代谢途径进而利用基因工程手段提升目的物质的产量来解决京大戟药源短缺问题具有重要意义。该研究以大戟愈伤组织为材料,使用茉莉酸甲酯分别按时间梯度和浓度梯度进行诱导,将诱导后的愈伤组织分为两部分:一部分提取其总RNA,以actin为内参基因进行反转录,实时定量RT-PCR分析大戟三萜类代谢途径中hmgr、sqs与fps基因的相对表达差异;另一部分用于提取其总三萜并使用分光光度法进行含量测定。实时定量RT-PCR分析结果表明,茉莉酸甲酯可诱导3个基因的表达,但其表达模式不一样。相应的京大戟愈伤组织中总三萜的含量明显提高,最高可较未处理样品增加27%。研究结果可为茉莉酸甲酯促进药用植物大戟三萜类物质积累的分子机制研究提供参考。     

外源茉莉酸甲酯对辣椒烟粉虱的影响

化学农药仍是目前蔬菜烟粉虱Bemisia tabaci防控的重要措施,研发有效的非化学措施防控烟粉虱是蔬菜绿色生产亟待解决的问题。本文以苏椒15号（感性品种）和新苏椒五号（抗性品种）辣椒Capsicum annuum L.为对象,通过在辣椒上喷施茉莉酸甲酯,研究外源茉莉酸甲酯对烟粉虱发育历期、存活率、消化酶活性和田间烟粉虱种群动态的影响。研究发现,取食茉莉酸甲酯处理的辣椒后烟粉虱的发育历期显著延长、存活率明显下降。其中取食感性品种辣椒和抗性品种辣椒的烟粉虱发育历期分别延长7.66 d和9.66 d,存活率分别下降10.5%和9.89%;取食感性品种辣椒的烟粉虱主要是延长卵期、4龄若虫期和伪蛹期,而取食抗性品种辣椒的烟粉虱各历期均延长。外源茉莉酸甲酯对烟粉虱体内消化酶的活性也有明显的影响,取食茉莉酸甲酯处理的辣椒后烟粉虱的消化酶活性显著下降,取食感性品种辣椒和抗性品种辣椒24 h后,胰蛋白酶活力分别较对照下降了61.86%和45.0%,糜蛋白酶活性分别较对照下降了47.29%和40.11%。大田辣椒上喷施茉莉酸甲酯后,烟粉虱的种群数量迅速下降,但在抗性品种和感性品种上烟粉虱的校正虫口减退率没有明显的差异。研究发现,外源茉莉酸甲酯处理,可以延缓烟粉虱的发育速率,降低胰蛋白酶和糜蛋白酶的活性,对田间烟粉虱种群也有较好的控制作用。     

Diel variability of mercury phase and species distributions in the Florida Everglades

Preliminary studies of mercury (Hg) cycling in the Everglades revealed that dissolved gaseous mercury (DGM), total mercury (Hg_T), and reactive mercury (Hg_R) show reproducible, diel trends. Peak water-column DGM concentrations were observed on or about noon, with a 3 to 7 fold increase over night-time concentrations. Production of DGM appears to cease during dark periods, with nearly constant water column concentrations that were at or near saturation with respect to the overlying air. A simple mass balance shows that the flux of Hg to the atmosphere from diel DGM production and evasion represents about 10% of the annual input from atmospheric deposition. Production of DGM is likely the result of an indirect photolysis reaction that involves the production of reductive species and/or reduction by electron transfer. Diel variability in Hg_T and Hg_R appears to be controlled by two factors: inputs from rainfall and photolytic sorption/desorption processes. A possible mechanism involves photolysis of chromophores on the surface of a solid substrate (e.g., the periphyton mat) giving rise to destabilization of sorbed mercury and net desorption during daylight. At night, the sorption reactions predominate and the water-column Hg_T decreases. Methylmercury (MeHg) also showed diel trends in concentration but were not clearly linked to the solar cycle or rainfall at the study site.     

啤酒抗日光嗅味的研究

啤酒中的３－甲基－２－丁烯－１－硫醇是由光照产生的。减少光照或减少其胶驱物质的产生是降低啤酒日光嗅味的有效途径。酵母和核黄素在ＭＢＴ的形成过程中起到关键作用同,还原酒花苦味物质能从根本上解决啤酒的日光嗅味。