597 research outputs found

    An interpretation and reevaluation of dilsey

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    In the case of The Sound and the Fury, critics have focused upon Dilsey, the only major character in the novel who embodies the universal truths cited in Faulkner\u27s Stockholm address

    Acylation of glucosaminyl phosphatidylinositol revisited. Palmitoyl-CoA dependent palmitoylation of the inositol residue of a synthetic dioctanoyl glucosaminyl phosphatidylinositol by hamster membranes permits efficient mannosylation of the glucosamine residue

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    Two critical steps in the assembly of yeast and mammalian glycosylphosphatidylinositol (GPI) anchor precursors are palmitoylation of the inositol residue and mannosylation of the glucosamine residue of the glucosaminyl phosphatidylinositol (GlcNα-PI) intermediate. Palmitoylation has been reported to be acyl-CoA dependent in yeast membranes (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 267, 8599-8603) but strictly acyl- CoA independent in rodent membranes (Stevens, V. L., and Zhang, H. (1994) J. Biol. Chem. 269, 31397-31403), and thus poorly conserved. In addition, it was suggested that acylation must precede mannosylation in both yeast (Costello, L. C., and Orlean, P. (1992) J. Biol. Chem. 276, 8599-8603) and rodent (Urakaze, M., Kamitani, T., DeGasperi, R., Sugiyama, E., Chang, H.-M., Warren, C. D., and Yeh, E. T. H. (1992) J. Biol. Chem. 267, 6459-6462) cells because GlcNα-acyl-PI accumulates in vivo when mannosylation is blocked. However, GlcNα-acyl-PI accumulation would also be expected if mannosylation and acylation were independent of each other. These issues were addressed by the use of a synthetic dioctanoyl GlcNα-PI analogue (GlcNα-PI(C8)) as an in vitro substrate for GPI-synthesizing enzymes in Chinese hamster ovary cell membranes. GlcNα-PI(C8) was acylated in an manner requiring acyl-CoA. Thus, the process involving acyl-CoA reported for yeast has been conserved in mammals. Furthermore, both GlcNα-PI(C8) and GlcNα-acyl-PI(C8) could be mannosylated in vitro, but mannosylation of the latter was significantly more efficient. This provides direct support for the earlier suggestion that acylation precedes mannosylation in rodents cells. A similar result was also observed with the Saccharomyces cerevisiae mannosyltransferase. In contrast, it has been reported that mannosylation of endogenous GlcNα-PI by Trypansoma brucei membranes occurs without prior acylation. The same result was obtained with GlcNα-PI(C8), confirming that the mannosyltransferase of trypanosomes is divergent from those in yeasts and rodents

    Working with simple machines

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    A set of examples is provided that illustrate the use of work as applied to simple machines. The ramp, pulley, lever and hydraulic press are common experiences in the life of a student and their theoretical analysis therefore makes the abstract concept of work more real. The mechanical advantage of each of these systems is also discussed so that students can evaluate their usefulness as machines.Comment: 9 pages, 4 figure

    Translation attenuation by PERK balances ER glycoprotein synthesis with lipid-linked oligosaccharide flux

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    Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc3Man9GlcNAc2 (G3M9Gn2) from the lipid-linked oligosaccharide (LLO) glucose3mannose9N-acetylglucosamine2-P-P-dolichol (G3M9Gn2-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2α (eIF2α) on Ser51 by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G3M9Gn2-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2α Ser51 phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser51 kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway

    A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults.

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    Strategies to enhance the immunogenicity of DNA vaccines in humans include i) co-administration of molecular adjuvants, ii) intramuscular administration followed by in vivo electroporation (IM/EP) and/or iii) boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study.Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG) plasmid DNA (pDNA) vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12) (GENEVAX IL-12) given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM.All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs) were reported. T cell and antibody response rates after HIVMAG (x3) prime-Ad35 (x1) boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ) ELISPOT responses was highest after HIVMAG (x3) without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS) were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3) prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected.The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected.ClinicalTrials.gov NCT01496989

    Mannose-6-phosphate regulates destruction of lipid-linked oligosaccharides.

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    Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses.This work is supported by NIH grants DK-042394, HL-052173, and HL-057346 to R.J.K.; by NIH grants AI-073898 and GM-056927 to I.M.; by NIH grant R-37-DK047119 and a Principal Research Fellowship from the Wellcome Trust to D.R.; by NIH grant GM-031278 and support from the Robert Welch Foundation to J.R.F.; and by NIH grant GM-038545 and Robert Welch Foundation grant I-1168 to M.A.L

    The introduction of modern physics: overcoming a deformed vision of science

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    In this paper, we try to show initially that modern physics is usually introduced in high school curricula without reference to the difficulties of classical physics, simply juxtaposing the two paradigms or even mixing them up. As a result, serious misconceptions arise. We then present another way of introducing modern physics, based on a contructivist view of science learning, and give some results obtained with the new materials

    Potential effects of oilseed rape expressing oryzacystatin-1 (OC-1) and of purified insecticidal proteins on larvae of the solitary bee Osmia bicornis

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    Despite their importance as pollinators in crops and wild plants, solitary bees have not previously been included in non-target testing of insect-resistant transgenic crop plants. Larvae of many solitary bees feed almost exclusively on pollen and thus could be highly exposed to transgene products expressed in the pollen. The potential effects of pollen from oilseed rape expressing the cysteine protease inhibitor oryzacystatin-1 (OC-1) were investigated on larvae of the solitary bee Osmia bicornis (= O. rufa). Furthermore, recombinant OC-1 (rOC-1), the Bt toxin Cry1Ab and the snowdrop lectin Galanthus nivalis agglutinin (GNA) were evaluated for effects on the life history parameters of this important pollinator. Pollen provisions from transgenic OC-1 oilseed rape did not affect overall development. Similarly, high doses of rOC-1 and Cry1Ab as well as a low dose of GNA failed to cause any significant effects. However, a high dose of GNA (0.1%) in the larval diet resulted in significantly increased development time and reduced efficiency in conversion of pollen food into larval body weight. Our results suggest that OC-1 and Cry1Ab expressing transgenic crops would pose a negligible risk for O. bicornis larvae, whereas GNA expressing plants could cause detrimental effects, but only if bees were exposed to high levels of the protein. The described bioassay with bee brood is not only suitable for early tier non-target tests of transgenic plants, but also has broader applicability to other crop protection products

    Applications of aerospace technology

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    Highlights are presented for the Research Triangle Institute (RTI) Applications Team activities over the past quarter. Progress in fulfilling the requirements of the contract is summarized, along with the status of the eight add-on tasks. New problem statements are presented. Transfer activities for ongoing projects with the NASA Centers are included

    Oligosaccharyltransferase Inhibition Induces Senescence in RTK-Driven Tumor Cells

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    Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high throughput screen and lead compound optimization campaign that delivered a cell permeable inhibitor (NGI-1). NGI-1 targets the oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small cell lung cancer cells NGI-1 blocks cell surface localization and signaling of the EGFR glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or FGFR) for survival. In these cell lines OST inhibition causes cell cycle arrest accompanied by induction of p21, autofluorescence, and changes in cell morphology; all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor tyrosine kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells
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