Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A₂ (PLA₂) and secretory PLA₂. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA₂ can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.     

云南红豆杉紫杉烷2α-O-苯甲酰转运酶基因的克隆及定性

通过RACE技术,克隆了云南红豆杉紫杉烷2α-O-苯甲酰转运酶基因(TyTBT),该酶催化2-去苯甲酰-7,13-二乙酰巴卡亭Ⅲ生成7,13-二乙酰巴卡亭Ⅲ,是紫杉醇合成途径中的关键酶之一.TyTBT基因cDNA全长1481 bp,含有1 320 bp的开放读码框,编码440个氨基酸的多肽,分子量为50 050 Da,等电点为6.17.氨基酸序列比对表明TyTBT同植物酰化酶家族的其它成员有较高的相似性,超过67%,同东北红豆杉和曼地亚红豆杉的紫杉烷2α-O-苯甲酰转运酶氨基酸序列的一致性和相似性达到最高,分别为95%和96%.广泛地比对分析证明这种较高的相似性在红豆杉属的其它酶家族中具有普遍性,进化树分析表明同东北红豆杉和曼地亚红豆杉的紫杉烷2α-O-苯甲酰转运酶(TBT)的相似性高于紫杉醇合成途径中的其它酰化酶.     

A novel RGD-toxin protein,Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC₅₀ value at 5.277 μM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.     

Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars

Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability.     

Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1

Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr(31), Thr(51), Thr(129) and Thr(143). Substitution of Thr(31) with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr(51), Thr(129) or Thr(143) with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr(31) is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr(31). Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.     

不同培养条件对猪卵母细胞IVM、IVF的影响

通过优化猪卵母细胞体外成熟、体外受精和胚胎体外发育体系,以进一步提高体外胚胎的生产效率和质量.研究了激素存在时间、不同激素和不同血清对猪卵母细胞体外成熟的影响;共培养体系、精卵作用时间、去除卵丘细胞的方法对猪体外受精及早期胚胎发育的影响.猪卵母细胞IVM培养48h,前24h内加入PMSG、hCG,后24h将其去除,卵母细胞总成熟率为79.54%;培养液添加15?S或15%NCS,卵母细胞成熟率分别为79.48%和74.81%;PMSG、HCG和E2配合使用后卵母细胞成熟率为81.42%.在IVF前用吹打法获得的卵裂率、桑椹胚率分别为37.89%和8.54%,精卵共孵育6h或8h的卵裂率(40.52%,37.24%)、桑椹胚率(8.42%,7.85%),以及用输卵管上皮细胞共培养所获得的卵裂率(40.84%)、桑椹胚率(9.53%)均显著高于其它各组.     

数据噪音构建基因布尔网络模型的方法

在分析基因数据时,往往有噪音出现,因此借用基因表达谱数据中的噪音来建立卡诺图,可以得到布尔网络逻辑函数。而且利用此方法确定蛋白质与蛋白质之间的逻辑关系,建立蛋白质的逻辑网络。通过该方法可以寻找直系同源簇蛋白质数据的逻辑关系。