StyS激酶自磷酸化对Pseudomonas putida B3中靛蓝生物合成的调节作用

在恶臭假单胞菌Pseudomonas putida B3中,靛蓝生物合成关键酶基因styAB上游存在一个二元调控系统StyS-StyR。StyS蛋白属于激酶家族,是磷酸化信号转导的重要媒介,但激酶StyS的自磷酸化传导机制对靛蓝生物合成的调节作用尚未探明,因此,研究以Pseudomonas putida B3中激酶蛋白StyS作为研究对象,发现了两个磷酸化结合位点,构建了磷酸化位点突变株。通过分析野生菌株P. putida B3、styS基因缺失菌株P. putida B3-ΔstyS、styS基因回补菌株P. putida B3-ΔstyS-8和磷酸化位点突变株P. putida B3-ΔstyS-1之间的靛蓝产量及酶活发现,P. putida B3产量为10.14mg/L,P. putida B3-ΔstyS-8产量为3.63mg/L,磷酸化位点突变菌株无激酶活性且失去了产生靛蓝的能力。结果表明,StyS自磷酸化作用在靛蓝发酵体系中起重要作用。研究结果将为靛蓝生物合成途径的进一步研究提供参考。     

Serine/threonine protein phosphatases in DNA damage response

DNA damage response (DDR) is among the most important of the mechanisms that maintain genome stability which, when destabilized, predisposes organs to cancer. Reversible phosphorylation mediated by protein kinases and protein phosphatases regulates most, if not all, cellular activities, including DDR. Protein kinase inhibitors have become the main focus of targeted therapy and anticancer drug development. However, our limited knowledge of protein phosphatase function is compromising our capacity to develop therapeutic agents against phosphatases. In this review, we summarize the roles of serine/threonine protein phosphatases involved in DDR and propose that in situ dephosphorylation of phosphoproteins by protein phosphatases, instead of proteasome-mediated degradation of phosphoproteins, is mainly employed by cells.     

Cytoskeleton reorganization and FAK phosphorylation are involved in lanthanum(Ⅲ)-promoted proliferation and differentiation in rat osteoblasts

The effect of lanthanum ion(La3+)on osteoblast function and cytoskeleton is assessed in vitro. Osteoblasts were isolated from Snraeuo-tjawiey tetai neonatal rats.Cell proliferation and gene expression levels of cbfa-1,alkaline phosphatase(ALP),osteocalcin(OC),bone sialo-protein(BSP)and osteopontin(OPN)were examined by cell counting and RT-PCR.Cytoskeleton F-actin was stained with rhodamine-con-jugated phalloidin and was visualized by a confocal microscope.As the results,10-8-10-4M La3+-induced osteoblast proliferation on day2.Data from the RT-PCR assay revealed that 10-6 M La3+ up-regulated the expression levels of ALP,BSP,and cbfa-lon day 4,while it enhanced the expressions of OC and OPN on day 21.The F-actin cytoskeleton was strengthened and reorganized under the exposure of La3+.In addition,the phosphorylation of focal adhesion kinase(FAK)was significantly promoted in 24 h evaluated by Western blot analysis.These findings indi-sate that La 3+ promotes osteoblast activity through the phosphorylation of F AK and reorganization of the cytoskeleton.     

含氧化膦结构半芳香聚酰胺的合成与表征

以含磷芳香二胺双(3–氨基苯基)苯基氧化膦(BAPPO)和己二酸为单体,通过Yamazaki膦酰化反应制备新型半芳香聚酰胺(PA6I).研究反应温度、单体浓度、溶剂体系以及反应时间对聚合物特性黏度的影响,得到特性黏度为0.47,dL/g的聚合物.利用傅里叶变换红外光谱(FTIR)、核磁共振谱(1H,NMR)对含磷半芳香聚酰胺进行结构表征;利用差示扫描量热法(DSC)和热重分析(TGA)研究新型半芳香聚酰胺的热性能.结果表明聚合物具有优良的热性能,Tg为206,℃,5%热分解温度为388.1,℃.薄膜样品的极限氧指数为43%,表明该聚合物有优良的阻燃性.     

4∶5,7∶8-二异亚丙基-3-去氧-D-甘露-2-辛酮酸(Kdo)甲酯及其对甲基硫苷合成

以D-甘露糖和L-酒石酸二甲酯为原料合成4∶5,7∶8-O-二异亚丙基-3-去氧-D-甘露-2-辛酮酸甲酯及其对甲基苯硫苷衍生物。首先,D-甘露糖通过缩酮反应制得2∶3,5∶6-O-二异亚丙基-D-甘露糖1;然后,L-酒石酸二甲酯2经过氧化水解,磷酰化和硅烷基化制得磷酸酯4。化合物1和磷酸酯4经Wittig-Horner反应缩合得到化合物5,脱除硅基制得4∶5,7∶8-二异亚丙基-3-去氧-β-D-甘露-2-辛酮酸甲酯6,总收率为34%。化合物6通过端基乙酰化和硫取代反应得到硫苷衍生物8,六步总收率为26%。产物结构经NMR和HRMS确证。     

维生素E对CA诱导的tau蛋白过度磷酸化的保护

通过免疫印迹，酶活性测定等方法在脑片水平研究维生素E(VE)对花萼海绵诱癌素(cA)诱导的骨架蛋白tau过度磷酸化的保护作用及机制．结果显示：01tmol／LCA使tau蛋白在Ser396／404，Serl98／199／202位点的磷酸化程度显著升高，丙二醛(MDA)含量及超氧化物歧化酶(s0D)活性显著升高；VE能使tau蛋白上述位点的磷酸化水平显著降低，同时降低CA诱导的vH)A含量的升高及进一步升高SK)D的活性．提示CA不仅能够在脑片水平诱导tau的过度磷酸化，同时诱导氧化应激反应；vE对CA诱导的氧化应激反应及tau蛋白过度磷酸化具有保护作用．     

OA诱导大鼠基底核Ach降低及τ蛋白过度磷酸化

研究了磷酸酯酶(proteinphosphatase，PP)对胆碱能神经元功能的影响，将PP抑制剂岗田酸(okadaic acid，OA)注入大鼠双侧Meynert基底核，并经免疫印迹检测r(tau)蛋白磷酸化程度、经微渗透结合HL,PC检测Ach、经Morris水迷宫检测大鼠的空间记忆能力，结果发现，Meynert基底核注射OA后，Ach水平降低，τ蛋白在Sei-198／Sei-199／Set-202，Ser-396／Ser-404位点发生过度磷酸化，并伴有大鼠空间记忆障碍，本研究结果提示PP活性降低可能参与了神经元纤维缠结形成、胆碱能神经元功能及认知功能障碍，在AD发病机制中起重要作用。     

Alteration of mitochondrial bioenergetics due to intravenous injection of a perfluorocarbon emulsion

Wistar albino rats were intravenously injected with 1 ml of an oxyphoretic emulsion of perfluorobutylfurane and killed 3, 7 or 30 days later. Mitochondria isolated from the liver and kidneys of treated rats showed a small decrease in the transmembrane electrical potential and a substantial depression of the rates of both ATP synthesis and ADP-stimulated respiration. These alterations in mitochondrial oxidative phosphorylation appear to be induced by perfluorocarbon and/or tensioactive molecules interacting with hydrophobic cell structures.     

Phytanic acid impairs mitochondrial respiration through protonophoric action

Refsum disease is a rare, inherited neurodegenerative disorder characterized by accumulation of the dietary branched-chain fatty acid phytanic acid in plasma and tissues caused by a defect in the alphaoxidation pathway. The accumulation of phytanic acid is believed to be the main pathophysiological cause of the disease. However, the exact mechanism(s) by which phytanic acid exerts its toxicity have not been resolved. In this study, the effect of phytanic acid on mitochondrial respiration was investigated. The results show that in digitonin-permeabilized fibroblasts, phytanic acid decreases ATP synthesis, whereas substrate oxidation per se is not affected. Importantly, studies in intact fibroblasts revealed that phytanic acid decreases both the mitochondrial membrane potential and NAD(P)H autofluorescence. Taken together, the results described here show that unesterified phytanic acid exerts its toxic effect mainly through its protonophoric action, at least in human skin fibroblasts. Received 4 August 2007; received after revision 26 September 2007; accepted 10 October 2007 J. C. Komen, F. Distelmaier: These authors contributed equally to this work.