An exceptional horizontal gene transfer in plastids: gene replacement by a distant bacterial paralog and evidence that haptophyte and cryptophyte plastids are sisters

Background  

Horizontal gene transfer (HGT) to the plant mitochondrial genome has recently been shown to occur at a surprisingly high rate; however, little evidence has been found for HGT to the plastid genome, despite extensive sequencing. In this study, we analyzed all genes from sequenced plastid genomes to unearth any neglected cases of HGT and to obtain a measure of the overall extent of HGT to the plastid.     

Tocopherol Transfer Protein Sensitizes Prostate Cancer Cells to Vitamin E

Prostate cancer is a major cause of mortality in men in developed countries. It has been reported that the naturally occurring antioxidant α-tocopherol (vitamin E) attenuates prostate cancer cell proliferation in cultured cells and mouse models. We hypothesized that overexpression of the tocopherol transfer protein (TTP), a vitamin E-binding protein that regulates tocopherol status, will sensitize prostate cancer cells to the anti-proliferative actions of the vitamin. To test this notion, we manipulated the expression levels of TTP in cultured prostate cells (LNCaP, PC3, DU145, and RWPE-1) using overexpression and knockdown approaches. Treatment of cells with tocopherol caused a time- and dose-dependent inhibition of cell proliferation. Overexpression of TTP dramatically sensitized the cells to the apoptotic effects of α-tocopherol, whereas reduction (“knockdown”) of TTP expression resulted in resistance to the vitamin. TTP levels also augmented the inhibitory effects of vitamin E on proliferation in semi-solid medium. The sensitizing effects of TTP were paralleled by changes in the intracellular accumulation of a fluorescent analog of vitamin E and by a reduction in intracellular levels of reactive oxygen species and were not observed when a naturally occurring, ligand binding-defective mutant of TTP was used. We conclude that TTP sensitizes prostate cancer cells to the anti-proliferative effects of vitamin E and that this activity stems from the ability of protein to increase the intracellular accumulation of the antioxidant. These observations support the notion that individual changes in the expression level or activity of TTP may determine the responsiveness of prostate cancer patients to intervention strategies that utilize vitamin E.     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

Linalool Dehydratase-Isomerase,a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V_max) were 140 nanokatals mg⁻¹ for the linalool dehydratase and 410 nanokatals mg⁻¹ for the geraniol isomerase, with apparent K_m values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).     

Shape similarity measures for the design of small RNA switches

Conformational switching in the secondary structure of RNAs has recently attracted considerable attention, fostered by the discovery of 'riboswitches' in living organisms. These are genetic control elements that were found in bacteria and offer a unique regulation mechanism based on switching between two highly stable states, separated by an energy barrier between them. In riboswitches, the energy barrier is crossed by direct metabolite binding, which facilitates regulation by allosteric means. However, other event triggers can cause switching to occur, such as single-point mutations and slight variations in temperature. Examples of switches with these event triggers have already been reported experimentally in the past. Here, the goal is to computationally design small RNA switches that rely on these triggers. Towards this end, our computer simulations utilize a variety of different similarity measures to assess the distances between an initial state and triggered states, based on the topology of the secondary structure itself. We describe these combined similarity measures that rely on both coarse-grained and fine-grained graph representations of the RNA secondary structure. As a result of our simulations, we provide some candidate sequences of approximately 30-50 nt, along with the exact triggers that drive the switching. The event triggers under consideration can be modelled by Zuker's mfold or the Vienna package. The proposed methodology that rely on shape measures can further be used to computationally generate more candidates by simulating various event triggers and calculating their effect on the shape.     

Inhibition of arginase I activity by RNA interference attenuates IL-13-induced airways hyperresponsiveness

Increased arginase I activity is associated with allergic disorders such as asthma. How arginase I contributes to and is regulated by allergic inflammatory processes remains unknown. CD4+ Th2 lymphocytes (Th2 cells) and IL-13 are two crucial immune regulators that use STAT6-dependent pathways to induce allergic airways inflammation and enhanced airways responsiveness to spasmogens (airways hyperresponsiveness (AHR)). This pathway is also used to activate arginase I in isolated cells and in hepatic infection with helminths. In the present study, we show that arginase I expression is also regulated in the lung in a STAT6-dependent manner by Th2-induced allergic inflammation or by IL-13 alone. IL-13-induced expression of arginase I correlated directly with increased synthesis of urea and with reduced synthesis of NO. Expression of arginase I, but not eosinophilia or mucus hypersecretion, temporally correlated with the development, persistence, and resolution of IL-13-induced AHR. Pharmacological supplementation with l-arginine or with NO donors amplified or attenuated IL-13-induced AHR, respectively. Moreover, inducing loss of function of arginase I specifically in the lung by using RNA interference abrogated the development of IL-13-induced AHR. These data suggest an important role for metabolism of l-arginine by arginase I in the modulation of IL-13-induced AHR and identify a potential pathway distal to cytokine receptor interactions for the control of IL-13-mediated bronchoconstriction in asthma.     

Swelling-activated K+ efflux and regulatory volume decrease efficiency in human bronchial epithelial cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.     

Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.     

Resistance Gene Analog Polymorphism (RGAP) Markers Co-Localize with Disease Resistance Genes and QTL in Common Bean

Resistance (R) genes containing nucleotide-binding site (NBS)-leucine rich repeats (LRR) are the most prevalent types of R gene in plants. The objective of this study was to develop PCR-based R-gene analog polymorphism (RGAP) markers for common bean (Phaseolus vulgaris L). Twenty degenerate primers were designed from the conserved kinase-1a (GVGKTT) and hydrophobic domains (GLPLAL) of known NBS-LRR type R-genes and from EST databases. Sixty-six of the 100 primer combinations tested yielded polymorphism. Thirty-two RGAP markers were mapped in the BAT 93/Jalo EEP558 core mapping population for common bean. The markers mapped to 10 of 11 linkage groups with a strong tendency for clustering. In addition, the RGAP markers co-located, on six linkage groups, with 15 resistance gene analogs (RGAs) that were previously mapped in other populations of common bean. The distance between the priming sites in NBS-LRR type R-genes is around 500 bp. Of the 32 RGAP markers, 19 had sizes larger and 13 less than 500 bp. RGAP markers mapped close to known R-genes on B11, and to QTLs for resistance on B1, B2, B6, B7, B8, B10, and B11. RGAP appears to provide a useful marker technique for tagging and mapping R-genes in segregating common bean populations, discovery of candidate genes underlying resistance QTL, and future cloning of R-genes in common bean.