Liaisons between Survivin and Plk1 during Cell Division and Cell Death

Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser²⁰ by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.     

Sheep-urine-induced changes in soil microbial community structure

Soil microbial communities play an important role in nutrient cycling and nutrient availability, especially in unimproved soils. In grazed pastures, sheep urine causes local changes in nutrient concentration which may be a source of heterogeneity in microbial community structure. In the present study, we investigated the effects of synthetic urine on soil microbial community structure, using physiological (community level physiological profiling, CLPP), biochemical (phospholipid fatty acid analysis, PLFA) and molecular (denaturing gradient gel electrophoresis, DGGE) fingerprinting methods. PLFA data suggested that synthetic urine treatment had no significant effect on total microbial (total PLFA), total bacterial or fungal biomass; however, significant changes in microbial community structure were observed with both PLFA and DGGE data. PLFA data suggested that synthetic urine induced a shift towards communities with higher concentrations of branched fatty acids. DGGE banding patterns derived from control and treated soils differed, due to a higher proportion of DNA sequences migrating only to the upper regions of the gel in synthetic urine-treated samples. The shifts in community structure measured by PLFA and DGGE were significantly correlated with one another, suggesting that both datasets reflected the same changes in microbial communities. Synthetic urine treatment preferentially stimulated the use of rhizosphere-C in sole-carbon-source utilisation profiles. The changes caused by synthetic urine addition accounted for only 10-15% of the total variability in community structure, suggesting that overall microbial community structure was reasonably stable and that changes were confined to a small proportion of the communities.     

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ~7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ~25 min as follows: (i) DNA immobilization ~6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ~6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light.     

Diverse functional motifs within the three intracellular loops of the CGRP1 receptor

The CGRP(1) receptor exists as a heterodimeric complex between a single-pass transmembrane accessory protein (RAMP1) and a family B G-protein-coupled receptor (GPCR) called the calcitonin receptor-like receptor (CLR). This study investigated the structural motifs found in the intracellular loops (ICLs) of this receptor. Molecular modeling was used to predict active and inactive conformations of each ICL. Conserved residues were altered to alanine by site-directed mutagenesis. cAMP accumulation, cell-surface expression, agonist affinity, and CGRP-stimulated receptor internalization were characterized. Within ICL1, L147 and particularly R151 were important for coupling to G(s). R151 may interact directly with the G-protein, accessing it following conformational changes involving ICL2 and ICL3. At the proximal end of ICL3, I290 and L294, probably lying on the same face of an alpha helix, formed a G-protein coupling motif. The largest effects on coupling were observed with I290A; additionally, it reduced CGRP affinity and impaired internalization. I290 may interact with TM6 to stabilize the conformation of ICL3, but it could also interact directly with Gs. R314, at the distal end of ICL3, impaired G-protein coupling and to a lesser extent reduced CGRP affinity; it may stabilize the TM6-ICL3 junction by interacting with the polar headgroups of membrane phospholipids. Y215 and L214 in ICL2 are required for cell-surface expression; they form a microdomain with H216 which has the same function. This study reveals similarities between the activation of CLR and other GPCRs in the role of TM6 and ICL3 but shows that other conserved motifs differ in their function.     

Evaluating bronchodilator effects in chronic obstructive pulmonary disease using diffusion-weighted hyperpolarized helium-3 magnetic resonance imaging

The objective of this study was to evaluate the regional effects of bronchodilator administration in chronic obstructive pulmonary disease (COPD) using hyperpolarized helium-3 ((3)He) MRI apparent diffusion coefficient (ADC). Ten COPD ex-smokers provided written, informed consent and underwent diffusion-weighted, hyperpolarized (3)He MRI, spirometry, and plethysmography before and 25 ± 2 min after bronchodilator administration. Pre- and postsalbutamol whole-lung (WL) ADC maps were generated and registered together to identify the lung regions containing the (3)He signal at both time points, and mean ADC within those regions of interest (ROI) was determined for a measurement of previously ventilated ROI ADC (ADC(P)). Lung ROI with (3)He signal at both time points was used as a binary mask on postsalbutamol WL ADC maps to obtain an ADC measurement for newly ventilated ROI (ADC(N)). Postsalbutamol, no significant differences were detected in WL ADC (P = 0.516). There were no significant differences between ADC(N) and ADC(P) postsalbutamol (P = 1.00), suggesting that the ADC(N) lung regions were not more emphysematous than the lung ROI participating in ventilation before bronchodilator administration. Postsalbutamol, a statistically significant decrease in ADC(P) (P = 0.01) was detected, and there were significant differences between ADC(P) in the most anterior and most posterior image slices (P = 0.02), suggesting a reduction in regional gas trapping following bronchodilator administration. Regional evaluation of tissue microstructure using hyperpolarized (3)He MRI ADC provides insights into lung alterations that accompany improvements in regional (3)He gas distribution after bronchodilator administration.     

Performance linked to residence time distribution by a novel wool-based bioreactor for tertiary sewage treatment

Laboratory-scale experiments were carried out using up-flow 7 L Submerged Aerated Filter reactors packed with wool fibre or commercial plastic pall rings, Kaldnes, (70% by volume) support media for the tertiary treatment of sewage. The performance of the wool bioreactor was more consistent than that with Kaldnes medium, for both TOC removal (93%) and SS removal (90%). Both plastic and wool-packed bioreactors achieved complete nitrification at the load of about 0.4 kgCOD/m³/day. The sludge yield of the wool bioreactor was almost half that of the bioreactor with Kaldnes suggesting that wool could retain residual organics and particulates. The wool however was degraded and it was concluded that wool would have to be considered as additional sacrificial adsorption capacity rather than an alternative medium. The performance was linked to the residence time distribution studies and these changes in the wool structure. Biomass growth increased the retention of the tracer in the wool reactor by, it was suggested, exposing a greater surface area. Results from the plastic media on the other hand showed increased mixing possibly by increasing the mobility of the plastic. Aeration increased the mixing in both reactors, and patterns were in all cases predominantly well-mixed.     

Antioxidative capacity of phenolic compounds extracted from Lolium perenne and Lolium arundinaceum infected with Neotyphodium (Hypocreales: Clavicipitaceae)

Members of Neotyphodium endophytic fungi infecting Lolium perenne L. and Lolium arundinaceum Darb. alter the synthesis of several metabolites. In this study we determined the antioxidative capacity of phenolic compounds from L. perenne and L. arundinaceum infected with Neotyphodium lolii (Latch, Christensen et Samuels) and Neotyphodium coenophialum (Morgan-Jones et Gams) Glenn, Bacon et Hanlin, respectively. The antioxidant capacity was determined by measuring the scavenging capacity of aqueous methanolic extracts to the free-radical DPPH (2,2-diphenyl-1-picrylhydrazyl). L. perenne infected with ‘wild-type’ strain endophyte showed the highest scavenging capacity, whereas endophyte-free showed the least. Those infected with the ‘novel’ strains AR1 and AR37 showed intermediate capacities. L. arundinaceum infected with the ‘novel’ strain AR542 showed a lower scavenging capacity compared with endophyte-free counterparts, regardless of the L. arundinaceum germplasm. The endophyte-free Mediterranean and Continental L. arundinaceum showed a higher capacity to scavenge DPPH when compared with the endophyte-infected Mediterranean and Continental L. arundinaceum. These results suggest that the endophytic fungi alter the antioxidative capacity of the grasses.     

Endophytic fungus-vascular plant-insect interactions

Insect association with fungi has a long history. Theories dealing with the evolution of insect herbivory indicate that insects used microbes including fungi as their principal food materials before flowering plants evolved. Subtlety and the level of intricacy in the interactions between insects and fungi indicate symbiosis as the predominant ecological pattern. The nature of the symbiotic interaction that occurs between two organisms (the insect and the fungus), may be either mutualistic or parasitic, or between these two extremes. However, the triangular relationship involving three organisms, viz., an insect, a fungus, and a vascular plant is a relationship that is more complicated than what can be described as either mutualism or parasitism, and may represent facets of both. Recent research has revealed such a complex relationship in the vertically transmitted type-I endophytes living within agriculturally important grasses and the pestiferous insects that attack them. The intricacy of the association depends on the endophytic fungus-grass association and the insect present. Secondary compounds produced in the endophytic fungus-grass association can provide grasses with resistance to herbivores resulting in mutualistic relationship between the fungus and the plant that has negative consequences for herbivorous insects. The horizontally transmitted nongrass type-II endophytes are far less well studied and as such their ecological roles are not fully understood. This forum article explores the intricacy of dependence in such complex triangular relationships drawing from well-established examples from the fungi that live as endophytes in vascular plants and how they impact on the biology and evolution of free-living as well as concealed (e.g., gall-inducing, gall-inhabiting) insects. Recent developments with the inoculation of strains of type-I fungal endophytes into grasses and their commercialization are discussed, along with the possible roles the endophytic fungi play in the galls induced by the Cecidomyiidae (Diptera).     

The Vitis vinifera C-repeat binding protein 4 (VvCBF4) transcriptional factor enhances freezing tolerance in wine grape

Chilling and freezing can reduce significantly vine survival and fruit set in Vitis vinifera wine grape. To overcome such production losses, a recently identified grapevine C‐repeat binding factor (CBF) gene, VvCBF4, was overexpressed in grape vine cv. ‘Freedom’ and found to improve freezing survival and reduced freezing‐induced electrolyte leakage by up to 2 °C in non‐cold‐acclimated vines. In addition, overexpression of this transgene caused a reduced growth phenotype similar to that observed for CBF overexpression in Arabidopsis and other species. Both freezing tolerance and reduced growth phenotypes were manifested in a transgene dose‐dependent manner. To understand the mechanistic basis of VvCBF4 transgene action, one transgenic line (9–12) was genotyped using microarray‐based mRNA expression profiling. Forty‐seven and 12 genes were identified in unstressed transgenic shoots with either a >1.5‐fold increase or decrease in mRNA abundance, respectively. Comparison of mRNA changes with characterized CBF regulons in woody and herbaceous species revealed partial overlaps, suggesting that CBF‐mediated cold acclimation responses are widely conserved. Putative VvCBF4‐regulon targets included genes with functions in cell wall structure, lipid metabolism, epicuticular wax formation and stress‐responses suggesting that the observed cold tolerance and dwarf phenotypes are the result of a complex network of diverse functional determinants.     

人微小病毒3个主要蛋白基因真核表达载体的构建

目的:分别克隆人细小病毒B19三个主要蛋白VP1、VP2、NS1全长基因,构建真核表达载体。方法:利用PCR和分子克隆技术,分别将B19病毒vp1、vp2、ns1基因全长片段扩增后,构建带荧光标签的真核表达载体;在人体细胞中表达并通过荧光、RT-PCR和Western Blot、测序等方法鉴定。结果:成功构建了包含B19病毒vp1、vp2、ns1全长基因,并在人体细胞中表达了VP1、VP2、NS1蛋白。结论:人微小病毒B19三个主要蛋白基因得到克隆和表达,为进行相关的研究奠定了基础。