Vimentin is important in the neural differentiation of PC12 cells promoted by sialylation

Sialic acid modification is a kind of post-translational modification. To investigate the regulation effect of sialic acid on neural differentiation, we used CycloManN propanyl perac (CycloManN pro), a metabolic precursor of sialic acid, to treat PC12 cells. We noted that CycloManN pro indeed robustly promoted global sialylation detected by MAL II lectin blot in PC12 cells. Simultaneously, we interestingly found that the neurite outgrowth of PC12 cells was significantly promoted by the CycloManN pro treatment. The profile analysis of sialylated proteins showed that a protein band at 55KD was greatly enhanced especially in PC12L cells after CycloManN pro treatment. After enrichment with lectin MAL II, the proteins in this band were analyzed by mass spectrometry. The results showed that 23 proteins were in the band, but the score of vimentin was the highest among them. To investigate further the role of vimentin in the process of neurite differentiation, vimentin construct was transfected into PC12 cells. We interestingly observed that ectopic expression of vimentin significantly enhanced the neurite outgrowth induced by CycloManN pro. However, after three potential glycosylation sites (Ser-7, Thr-33, Ser-34:) of vimentin were mutated to alanine, overexpression of the mutated vimentin completely lost the enhancement activity for the neural differentiation even in the presence of CycloManN pro. Taken together, our study demonstrated that vimentin was important in the induction of neural differentiation by CycloManN pro.     

Mammalian <Emphasis Type="Italic">CYP2E1</Emphasis> gene triggered changes of relative ions fluxes,CaM content and genes expression profiles in <Emphasis Type="Italic">Petunia hybrida</Emphasis> cells to enhance resistance to formaldehyde

The influence of light quality and cytokinin content in media on growth, development, photosynthetic pigments and secondary metabolite content of Myrtus communis L. was evaluated in an in vitro culture. Various treatments with light emitting diodes (LEDs): 100% blue (B), a mix of 70% red and 30% blue (RB) and 100% red were applied and compared with a traditional fluorescent lamp as control. Axillary shoots were incubated on Murashige and Skoog medium with 30 g dm^?3 sucrose, 0.5% BioAgar, 0.5 μM 1-naphthaleneacetic acid and different concentrations of 6-benzyladenine (BA): 1, 2.5 and 5 µM. Cultures were maintained for 6 weeks in 23/21?±?1 °C (day/night), 80% relative humidity and 16/8 h photoperiod; photosynthetic photon flux density (PPFD) was 35 µmol m^?2 s^?1 in all treatments. Light spectra and BA content in media affected biometrical and phytochemical M. communis properties. Red LEDs and 5 µM BA resulted in the highest multiplication rate. The highest shoots were obtained under red LEDs, but with the lowest concentration of cytokinin in media. Fresh weight was greatest on LEDs containing blue light in the spectrum (B and RB); moreover, 5 µM BA increased dry weight. Photosynthetic pigment levels were lower under LED light compared to control lamps. Phenolic acids and flavonoids were identified in M. communis leaf extracts. Myricetin was the major constituent with highest concentration under red LEDs and highest BA level.     

Proteasome Inhibition-Induced Downregulation of Akt/GSK-3β Pathway Contributes to Abnormality of Tau in Hippocampal Slice

Proteasome inhibition can induce abnormal accumulation and phosphorylation of microtubule-associated protein tau. The major function of tau protein is to promote microtubules assembly and stabilization, and abnormal tau protein would disturb its microtubule-binding function. In this study, proteasome inhibitor MG132 was used to treat hippocampal slices to explore the role and mechanism of Akt/glycogen synthase kinase-3β (GSK-3β) in proteasome inhibition-induced tau abnormality. During the culture period, we measure the lactate dehydrogenase (LDH) content to assay the viability of hippocampal slices. Following 2.5 and 5 μM MG132 treatment for 6 h, we detected the expression, phosphorylation modification, and microtubule-binding function of tau protein of slices. We also analyzed the changed activities of glycogen synthase kinase-3β (GSK-3β) and protein kinase B (PKB/Akt) and the level of heat shock protein 90 (Hsp90) in the process. In addition, co-immunoprecipitation was used to investigate the interaction between Akt and Hsp90, Akt and protein phosphatase-2A (PP2A) in the MG132-treated organotypic hippocampal slices. Our results indicated that proteasome inhibition led to degradation obstacles and abnormal phosphorylation of tau protein. The downregulated Akt/GSK-3β signaling pathway might be responsible for the abnormal phosphorylation of tau protein at multiple sites which further reduced the microtubule-binding function of tau protein. Furthermore, proteasome inhibition decreased the binding capacity of Akt-Hsp90 while increased the Akt-PP2A binding ability which mediated Akt inactivity. This current study establishes a hippocampal slice model targeting Akt/GSK-3β signaling pathway to explore the pivotal role of proteasome inhibition in tau pathology.     

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.     

Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development

Glycosaminoglycans (GAGs) play a central role in embryonic development by regulating the movement and signaling of morphogens. We have previously demonstrated that GAGs are the co-receptors for Fgf10 signaling in the lacrimal gland epithelium, but their function in the Fgf10-producing periocular mesenchyme is still poorly understood. In this study, we have generated a mesenchymal ablation of UDP-glucose dehydrogenase (Ugdh), an essential biosynthetic enzyme for GAGs. Although Fgf10 RNA is expressed normally in the periocular mesenchyme, Ugdh mutation leads to excessive dispersion of Fgf10 protein, which fails to elicit an FGF signaling response or budding morphogenesis in the presumptive lacrimal gland epithelium. This is supported by genetic rescue experiments in which the Ugdh lacrimal gland defect is ameliorated by constitutive Ras activation in the epithelium but not in the mesenchyme. We further show that lacrimal gland development requires the mesenchymal expression of the heparan sulfate N-sulfation genes Ndst1 and Ndst2 but not the 6-O and 2-O-sulfation genes Hs6st1, Hs6st2 and Hs2st. Taken together, these results demonstrate that mesenchymal GAG controls lacrimal gland induction by restricting the diffusion of Fgf10.     

Cell suspension culture-mediated incorporation of the rice bel gene into transgenic cotton

Cotton plants engineered for resistance to the herbicides, glyphosate or glufosinate have made a considerable impact on the production of the crop worldwide. In this work, embryogenic cell cultures derived from Gossypium hirsutum L. cv Coker 312 hypocotyl callus were transformed via Agrobacterium tumefaciens with the rice cytochrome P450 gene, CYP81A6 (bel). In rice, bel has been shown to confer resistance to both bentazon and sulfanylurea herbicides. Transformed cells were selected on a liquid medium supplemented alternately or simultaneously with kanamycin (50mg/L) and bentazon (4.2 μmol). A total of 17 transgenic cotton lines were recovered, based on the initial resistance to bentazon and on PCR detection of the bel transgene. Bel integration into the cotton genome was confirmed by Southern blot and expression of the transgene was verified by RT-PCR. In greenhouse and experimental plot trials, herbicide (bentazon) tolerance of up to 1250 mg/L was demonstrated in the transgenic plants. Transgenic lines with a single copy of the bel gene showed normal Mendelian inheritance of the characteristic. Importantly, resistance to bentazon was shown to be stably incorporated in the T1, T2 and T3 generations of self-fertilised descendents and in plants outcrossed to another upland cotton cultivar. Engineering resistance to bentazon in cotton through the heterologous expression of bel opens the possibility of incorporating this trait into elite cultivars, a strategy that would give growers a more flexible alternative to weed management in cotton crops.     

植物锌铁转运蛋白ZIP基因家族的研究进展

金属离子对植物的正常发育至关重要,但过量又会中毒.植物体内的自动调节平衡机制会调节金属离子的吸收和运输,从而控制金属离子的含量.锌铁调控蛋白ZIP( ZRT,IRT-like protein)家族是广泛存在于植物中的转运蛋白,具有Ca2+、Fe2+、Mn2+及Zn2+等多种金属元素的转运功能.了解ZIP转运体在植物中如何发挥离子转运功能,从分子水平认识金属离子缺乏或过量积累的机理有重要意义.综述拟南芥、水稻、大麦、苜蓿和玉米ZIP家族成员及其研究进展.     

Proteomic Analysis of High Temperature Stress-Responsive Proteins in Chrysanthemum Leaves

Chrysanthemum is one of the most important ornamental flowers in the world, and temperature has a significant influence on its field production. In the present study, differentially expressed proteins were investigated in the leaves of Dendranthema grandiflorum ‘Jinba’ under high temperature stress using label-free quantitative proteomics techniques. The expressed proteins were comparatively identified and analyzed. A total of 1,463 heat-related, differentially expressed proteins were successfully identified by Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS), and 1,463 heat-related, differentially expressed proteins were successfully identified by mass spectrometry after a high temperature treatment. Among these, 701 proteins were upregulated and 762 proteins were downregulated. The in-depth bioinformatics analysis of these differentially expressed proteins revealed that these were involved in energy metabolism pathways, protein metabolism, and heat shock. In the present study, the investigators determined the changes in the levels of some proteins, and their expression at the protein and molecular levels in chrysanthemum to help reveal the mechanism of heat resistance in chrysanthemum. Furthermore, the present study elucidated some of the proteins correlated to heat resistance in chrysanthemum, and their expression changes at the protein and molecular levels to help reveal the mechanism of heat resistance in this flower species. These results provide a theoretical basis for the selection of new heat resistant varieties of chrysanthemum in the field.     

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.     

用732阳离子交换树脂柱提取碱性氨基酸工艺研究

本文是从棉籽饼粕中,应用７３２阳离子树脂柱提取碱性氨基酸。研究结果表明,碱性氨基酸与中性氨基酸之间交叉部分较小。因此,本法即简化了提取工序,又缩短了操作时间,大大提高了氨基酸的分离效果。