翅碱蓬CMO基因克隆、测序及植物表达载体的构建

从翅碱蓬叶片中提取总RNA,根据相关同源序列设计引物,使用反转录试剂盒进行RT-PCR,扩增得到翅碱蓬胆碱单加氧酶（CMO）cDNA,进行PCR产物测序;然后将CMOcDNAT-A克隆至pMD18-T-simple载体上,经测序正确后亚克隆至pBI121植物表达载体,进行酶切鉴定及克隆测序。     

The role of Thr5 in human neuron growth inhibitory factor

GIF, a member of the metallothionein (MT) family (assigned as MT3), is a neuron growth inhibitory factor that inhibits neuron outgrowth in Alzheimer’s disease. The conserved Thr5 is one of the main differences between GIF and other members in the MT family. However, natural sheep GIF has an unusual Ala5, casting doubt on the role of common Thr5. We constructed a series of human GIF mutants at site 5, and characterized their biochemical properties by UV spectroscopy, circular dichroism spectroscopy, EDTA reaction, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) reaction, and pH titration. Their inhibitory activity toward neuron survival and neurite extension was also examined. Interestingly, the T5A mutant exhibited distinct metal thiolate activity in the EDTA and DTNB reactions, and also lost its bioactivity. Meanwhile, the T5S mutant had similar biochemical properties and biological activity as wild-type human GIF, indicating the hydroxyl group on the Thr5 was critical to the bioactivity of human GIF. We suggest the hydroxyl group in human GIF may help stabilize the biologically active conformation. On the other hand, lack of the hydroxyl group in sheep GIF may be partially compensated by its abnormal structure.Bin Cai and Qi Zheng contributed equally to this work.     

Attenuation of MPTP-induced neurotoxicity and locomotor dysfunction in Nucling-deficient mice via suppression of the apoptosome pathway

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is one of the experimental models most commonly used to study the pathogenesis of Parkinson's disease (PD). Although the biochemical mechanisms underlying the cell death induced by MPTP remain to be clarified, it has been found that the mitochondrial apoptotic signaling pathway plays an important role in the neurotoxicity of MPTP. Nucling is a novel type of apoptosis-associated molecule, essential for cytochrome c, apoptosis protease activating factor 1 (Apaf-1), pro-caspase-9 apoptosome induction and caspase-9 activation following pro-apoptotic stress. Here we found that Nucling-deficient mice treated with MPTP did not exhibit locomotor dysfunction in an open-field test. The substantia nigra dopaminergic neurons of Nucling-deficient mice were resistant to the damaging effects of the neurotoxin MPTP. Up-regulated expression of apoptosome was attenuated in Nucling-deficient mice treated with MPTP. These results indicate an important role for Nucling in MPTP-induced neuronal degeneration and suggest that the suppression of Nucling would be of therapeutic benefit for the treatment of neurodegeneration in PD.     

Oxaliplatin enhances TRAIL-induced apoptosis in gastric cancer cells by CBL-regulated death receptor redistribution in lipid rafts

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family that selectively induces apoptosis in cancer cells. However, gastric cancer cells are insensitive to TRAIL. In the present study, we show that oxaliplatin enhanced TRAIL-induced apoptosis of MGC803, BGC823, and SGC7901 cells. Oxaliplatin promoted death receptor 4 (DR4) and death receptor 5 (DR5) clustering into aggregated lipid rafts, while the cholesterol-sequestering agent nystatin partially prevented lipid raft aggregation, DR4 and DR5 clustering, and reduced apoptosis. Furthermore, the expression of the casitas B-lineage lymphoma (Cbl) family was downregulated by oxaliplatin. Transfection of c-Cbl or Cbl-b partially reversed oxaliplatin-induced lipid raft aggregation. These results indicated that oxaliplatin enhanced TRAIL-induced gastric cancer cell apoptosis at least partially through Cbl-regulated death receptor redistribution in lipid rafts.     

QTL analyses of seed weight during the development of soybean (Glycine max L. Merr.)

At harvest traits such as seed weight are the sum of development and responses to stresses over the growing season and particularly during the reproductive phase of growth. The aim here was to measure quantitative trait loci (QTL) underlying the seed weight from early development to drying post harvest. One hundred forty-three F(5) derived recombinant inbred lines (RILs) developed from the cross of soybean cultivars 'Charleston' and 'Dongnong 594' were used for the analysis of QTL underlying mean 100-seed weight at six different developmental stages. QTL x Environment interactions (QE) were analyzed by a mixed genetic mode based on 3 years' data. At an experiment-wise threshold of a=0.05 and by single-point analysis 94 QTL unaffected by QE underlay the mean seed weight at different developmental stages. Sixty-eight QTL affected by QE that also underlay mean seed weight were identified. From the 162 QTL 42 could be located on 12 linkage groups by composite interval mapping (LOD>2.0). The numbers, locations and types of the QTL and the genetic effects were different at each developmental stage. On linkage group C2 the distantly linked QTL swC2-1, swC2-2 and swC2-3 each affected mean seed weight throughout the different developmental stages. The DNA markers linked to the QTL possessed potential for use in marker-assisted selection for soybean seed size. The identification of QTL with genetic main effects and QE interaction effects suggested that such interactions might significantly alter seed weight during seed development.     

Placental TonEBP/NFAT5 osmolyte regulation in an ovine model of intrauterine growth restriction

TonEBP/NFAT5 (the tonicity-responsive enhancer binding protein/nuclear factor of activated T cells) modulates cellular response to osmotic changes by accumulating inositol and sorbitol inside the cells. Our objective was to assess placental osmolytes, TonEBP/NFAT5 RNA and protein expression, and signaling molecules across gestation between control and intrauterine growth restriction (IUGR) ovine pregnancies. Pregnant sheep were placed in hyperthermic conditions to induce IUGR. Placental tissues were collected at 55, 95, and 130 days gestational age (dGA) to measure inositol, sorbitol, TonEBP/NFAT5 (NFAT5), sodium-dependent myo-inositol transporter (SMIT; official symbol SLC5A3), aldose reductase (AR), and NADPH (official symbol DE-CR1). Placental weight was reduced in IUGR compared to controls at 95 and 130 dGA. Osmolyte concentrations were similar between control and IUGR placentas, but both groups demonstrated a significant decrease in inositol concentration and an increase in sorbitol concentration with advancing gestation. Cytosolic NFAT5 protein decreased significantly from 55 to 95 dGA in both groups, and nuclear NFAT5 protein increased only at 130 dGA in the IUGR group, but no differences were seen between groups for either cytosolic or nuclear NFAT5 protein concentrations. DE-CR1 concentrations were similar between groups and increased significantly with advancing gestational age. AR was lowest at 55dGA, and SLC5A3 increased with advancing gestational age. We conclude that both placental osmolytes inositol and sorbitol (and their corresponding proteins SLC5A3 and AR) change with gestational age and are regulated, at least in part, by NFAT5 and DE-CR1 (NADPH). The inverse relationship between each osmolyte across gestation (e.g., inositol higher in early gestation and sorbitol higher in late gestation) may reflect nutritional needs that change across gestation.     

Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1

Necrotic cell death is prevalent in many different pathological disease states and in traumatic injury. Necroptosis is a form of necrosis that stems from specific signaling pathways, with the key regulator being receptor interacting protein 1 (RIP1), a serine/threonine kinase. Specific inhibitors of RIP1, termed necrostatins, are potent inhibitors of necroptosis. Necrostatins are structurally distinct from one another yet still possess the ability to inhibit RIP1 kinase activity. To further understand the differences in the binding of the various necrostatins to RIP1 and to develop a robust high-throughput screening (HTS) assay, which can be used to identify new classes of RIP1 inhibitors, we synthesized fluorescein derivatives of Necrostatin-1 (Nec-1) and Nec-3. These compounds were used to establish a fluorescence polarization (FP) assay to directly measure the binding of necrostatins to RIP1 kinase. The fluorescein-labeled compounds are well suited for HTS because the assays have a dimethyl sulfoxide (DMSO) tolerance up to 5% and Z' scores of 0.62 (fluorescein-Nec-1) and 0.57 (fluorescein-Nec-3). In addition, results obtained from the FP assays and ligand docking studies provide insights into the putative binding sites of Nec-1, Nec-3, and Nec-4.     

Inhibitory effect of curcumin on the Al(III)-induced Aβ₄₂ aggregation and neurotoxicity in vitro

The pathogenesis of Alzheimer's disease (AD) involves a key event which changes the morphology of amyloid-β 42 (Aβ)?? peptide from its soluble monomeric form into the fibrillated aggregates in the brain. Aluminum ion, Al(III), is known to act as a pathological chaperone of the Aβ?? in this process; curcumin, a natural phenolic compound, is considered capable of binding Al(III) and Aβ??; nevertheless, little is known about the combined action of curcumin and Al(III) on the Aβ?? fibrillation and neurotoxicity. Here, combinations of circular dichroism spectroscopy, thioflavin T fluorescence, atomic force microscopy, Bradford and MTT assays, it is demonstrated that although Al(III) can promote the Aβ?? fibrillation dose-dependently, leading to the high neurotoxicity to PC12 cells, curcumin can inhibit the events. Besides, we found that curcumin is able not only to inhibit the formation of Al(III)-induced Aβ?? fibrillation, but also to form the Al(III)-curcumin complexes which in turn can remold the preformed, mature, ordered Aβ?? fibrils into the low toxic amorphous aggregates. These findings suggest that curcumin could block the binding of Al(III) with Aβ?? and form the Al(III)-curcumin complexes, so as to inhibit the Al(III)-induced Aβ?? fibrillation and neurotoxicity. The Al(III)-curcumin complexes are worth potentially developing as a therapy agent against the neurodegenerative disorders in the future.