74 research outputs found

    Ectopic Expression of Arabidopsis Phospholipase A Genes Elucidates Role of Phospholipase Bs in S. cerevisiae Cells

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    In S. cerevisiae neither disruption of the phospholipase B triple knockout mutant (plb1plb2plb3; plb123) nor over-expression of phospholipase Bs (PLBs) result in a phenotype different from wild type. In performing experiments to characterize candidate plant phospholipase (PLA) genes, we found, surprisingly, that ectopic expression of either of two different A. thaliana PLA2 or PLA1 genes in the yeast plb123 mutant completely inhibited cell growth. We proposed that while PLBs might not be essential for growth and metabolism of yeast cells, they may play an important role in cell survival by metabolizing excess intracellular lysophospholipids. To test our hypothesis, we overexpressed a plant phospholipase A2 (PLA2) in both WT and plb123 cells, producing a pool of lysophosphatidylcholine (lysoPtdCho) in both transformants. In 14C acetate labeling experiments, WT cells were able to catabolize the resultant labeled lysoPtdCho, preventing accumulation, and the cells grew normally. In contrast, in the triple plb123 mutant PLA2 transformant, lysoPtDCho accumulated more than 4-fold to a toxic level, inhibiting cell growth. However, this growth inhibition was complemented by co-expression of either PLB1, PLB2 or PLB3 in the plb123 triple mutant already expressing the plant PLA2. Furthermore, in labeling experiments, the rescued cells exhibited a 60-75% reduction in 14C-lysoPtdCho build-up compared to plb123PLA2 cells. Our data provides conclusive evidence that yeast PLBs can metabolize intracellular lysoPtdCho produced by plant PLA2 overexpression in yeast. Our experiments indicate the utility of ectopic plant phospholipase A gene expression to characterize poorly-understood phospholipid metabolism mutants in yeast or other organisms

    Cloning and functional characterization of the fatty acid elongase 1 (FAE1) gene from high erucic Crambe abyssinica cv. Prophet.

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    A genomic fatty acid elongation 1 (FAE1) clone was isolated from Crambe abyssinica. The genomic clone corresponds to a 1521-bp open reading frame, which encodes a protein of 507 amino acids. In yeast cells expression of CrFAE led to production of new very long chain monounsaturated fatty acids such as eicosenoic (20:1(delta11)) and erucic (22:1(delta13)) acids. Seed-specific expression in Arabidopsis thaliana resulted in up to a 12-fold increase in the proportion of erucic acid. On the other hand, in transgenic high-erucic Brassica carinata plants, the proportion of erucic acid was as high as 51.9% in the best transgenic line, a net increase of 40% compared to wild type. These results indicate that the CrFAE gene encodes a condensing enzyme involved in the biosynthesis of very long-chain fatty acids utilizing monounsaturated and saturated acyl substrates, with a strong capability for improving the erucic acid content

    Triacylglycerol synthesis by PDAT1 in the absence of DGAT1 activity is dependent on re-acylation of LPC by LPCAT2

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    <p>Abstract</p> <p>Background</p> <p>The <it>Arabidopsis thaliana dgat1 </it>mutant, <it>AS11</it>, has an oil content which is decreased by 30%, and a strongly increased ratio of 18:3/20:1, compared to wild type. Despite lacking a functional DGAT1, <it>AS11 </it>still manages to make 70% of WT seed oil levels. Recently, it was demonstrated that in the absence of <it>DGAT1</it>, <it>PDAT1 </it>was essential for normal seed development, and is a dominant determinant in <it>Arabidopsis </it>TAG biosynthesis.</p> <p>Methods</p> <p>Biochemical, metabolic and gene expression studies combined with genetic crossing of selected <it>Arabidopsis </it>mutants have been carried out to demonstrate the contribution of <it>Arabidopsis </it>PDAT1 and LPCAT2 in the absence of DGAT1 activity.</p> <p>Results</p> <p>Through microarray and RT-PCR gene expression analyses of <it>AS11 </it>vs. WT mid-developing siliques, we observed consistent trends between the two methods. <it>FAD2 </it>and <it>FAD3 </it>were up-regulated and <it>FAE1 </it>down-regulated, consistent with the <it>AS11 </it>acyl phenotype. <it>PDAT1 </it>expression was up-regulated by <it>ca </it>65% while <it>PDAT2 </it>expression was up-regulated only 15%, reinforcing the dominant role of <it>PDAT1 </it>in <it>AS11 </it>TAG biosynthesis. The expression of <it>LPCAT2 </it>was up-regulated by 50-75%, while <it>LPCAT1 </it>expression was not significantly affected. <it>In vitro </it>LPCAT activity was enhanced by 75-125% in microsomal protein preparations from mid-developing <it>AS11 </it>seed <it>vs </it>WT. Co-incident homozygous knockout lines of <it>dgat1</it>/<it>lpcat2 </it>exhibited severe penalties on TAG biosynthesis, delayed plant development and seed set, even with a functional PDAT1; the double mutant <it>dgat1/lpcat1 </it>showed only marginally lower oil content than <it>AS11</it>.</p> <p>Conclusions</p> <p>Collectively, the data strongly support that in <it>AS11 </it>it is <it>LPCAT2 </it>up-regulation which is primarily responsible for assisting in PDAT1-catalyzed TAG biosynthesis, maintaining a supply of PC as co-substrate to transfer <it>sn</it>-2 moieties to the <it>sn</it>-3 position of the enlarged <it>AS11 </it>DAG pool.</p

    Precession of a Freely Rotating Rigid Body. Inelastic Relaxation in the Vicinity of Poles

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    When a solid body is freely rotating at an angular velocity Ξ©{\bf \Omega}, the ellipsoid of constant angular momentum, in the space Ξ©1,Ξ©2,Ξ©3\Omega_1, \Omega_2, \Omega_3, has poles corresponding to spinning about the minimal-inertia and maximal-inertia axes. The first pole may be considered stable if we neglect the inner dissipation, but becomes unstable if the dissipation is taken into account. This happens because the bodies dissipate energy when they rotate about any axis different from principal. In the case of an oblate symmetrical body, the angular velocity describes a circular cone about the vector of (conserved) angular momentum. In the course of relaxation, the angle of this cone decreases, so that both the angular velocity and the maximal-inertia axis of the body align along the angular momentum. The generic case of an asymmetric body is far more involved. Even the symmetrical prolate body exhibits a sophisticated behaviour, because an infinitesimally small deviation of the body's shape from a rotational symmetry (i.e., a small difference between the largest and second largest moments of inertia) yields libration: the precession trajectory is not a circle but an ellipse. In this article we show that often the most effective internal dissipation takes place at twice the frequency of the body's precession. Applications to precessing asteroids, cosmic-dust alignment, and rotating satellites are discussed.Comment: 47 pages, 1 figur

    Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts

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    <p>Abstract</p> <p>Background</p> <p>Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of <it>DGAT1 </it>results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating <it>DGAT1 </it>levels during seed development, a concerted transcriptome and metabolome analysis of transgenic <it>B. napus </it>prototypes was performed.</p> <p>Results</p> <p>Using a targeted <it>Brassica </it>cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24–33% of the targets showing significant changes have no matching gene in <it>Arabidopsis </it>although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 <it>Arabidopsis </it>genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development.</p> <p>Conclusion</p> <p>Our results indicate that the increased TAG accumulation observed in transgenic <it>DGAT1 </it>plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in <it>Arabidopsis </it>compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.</p

    Handling Conflicts in Depth-First Search for LTL Tableau to Debug Compliance Based Languages

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    Providing adequate tools to tackle the problem of inconsistent compliance rules is a critical research topic. This problem is of paramount importance to achieve automatic support for early declarative design and to support evolution of rules in contract-based or service-based systems. In this paper we investigate the problem of extracting temporal unsatisfiable cores in order to detect the inconsistent part of a specification. We extend conflict-driven SAT-solver to provide a new conflict-driven depth-first-search solver for temporal logic. We use this solver to compute LTL unsatisfiable cores without re-exploring the history of the solver.Comment: In Proceedings FLACOS 2011, arXiv:1109.239

    Bacterial Heat-Stable Enterotoxins: Translation of Pathogenic Peptides into Novel Targeted Diagnostics and Therapeutics

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    Heat-stable toxins (STs) produced by enterotoxigenic bacteria cause endemic and traveler’s diarrhea by binding to and activating the intestinal receptor guanylyl cyclase C (GC-C). Advances in understanding the biology of GC-C have extended ST from a diarrheagenic peptide to a novel therapeutic agent. Here, we summarize the physiological and pathophysiological role of GC-C in fluid-electrolyte regulation and intestinal crypt-villus homeostasis, as well as describe translational opportunities offered by STs, reflecting the unique characteristics of GC-C, in treating irritable bowel syndrome and chronic constipation, and in preventing and treating colorectal cancer

    burst emission from the soft gamma repeater SGR 1900+14, Astrophys

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    Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. ABSTRACT We present evidence for burst emission from SGR 1900ϩ14 with a power-law high-energy spectrum extending beyond 500 keV. Unlike previous detections of high-energy photons during bursts from soft gamma repeaters (SGRs), these emissions are not associated with extraordinarily bright flares. Not only is the emission hard, but the spectra are better fitted by D. Band&apos;s gamma-ray burst (GRB) function rather than by the traditional optically thin thermal bremsstrahlung model. We find that the spectral evolution within these hard events obeys a hardness/ intensity anticorrelation. Temporally, these events are distinct from typical SGR burst emissions in that they are longer (∼1 s) and have relatively smooth profiles. Despite a difference in peak luminosity of տ10 11 between these bursts from SGR 1900ϩ14 and cosmological GRBs, there are striking temporal and spectral similarities between the two kinds of bursts, aside from spectral evolution. We outline an interpretation of these events in the context of the magnetar model
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