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Among the different vaccination approaches, DNA/RNA vaccination represents a promising means in particular for the induction of effective cellular immune responses conferred by CD8-positive T lymphocytes. To achieve such immune responses, there is a need for novel delivery systems that allow the introduction of nucleic acids to the cytosol of immune cells. We show, for the first time, the delivery of functional DNA and messenger RNA (mRNA) to mammalian antigen-presenting cells, including murine macrophages and human dendritic cells, using the yeast Saccharomyces cerevisiae as the delivery vehicle. After transfer of the particular nucleic acid, subsequent antigen processing and presentation were demonstrated in a human system. Remarkably, release of DNA/mRNA does not require additional ‘helper’ proteins such as listeriolysin. In conclusion, the yeast-based system described here is superior to many bacterial and viral systems in terms of efficacy, safety and targeting suggesting ‘mycofection’ as a promising approach for the development of a novel type of live vaccines.

Pyruvate phosphate dikinase (PPDK) catalyses the phosphorylation of pyruvate (+ ATP + Pi) yielding phosphoenolpyruvate (+ AMP + PPi). PPDK levels are induced upon drought and salt stress and both cytosolic and plastidic isoforms are found in higher plants. Herein we investigate the role of this enzyme upon aluminum exposure. We generated transgenic tobacco plants expressing either a plastidic (PPDK) or a putative cytosolic (ΔPPDK) Mesembryanthemum crystallinum PPDK under the transcriptional control of the patatin B33 promoter and found that they can ameliorate Al stress. Enhanced root growth in the presence of Al was observed in transgenic lines, while that of wild-type (WT) seedlings was strongly inhibited. Al content in root tips of stressed plants was reduced in the transgenic lines compared with WT, as judged by Eriochrome cyanine R (ECR) staining and atomic absorption spectrometry. Moreover, all transformants excreted higher levels of citric acid cycle intermediates (citrate and malate) than the WT seedlings did. Interestingly, we found different behavior between the two sets of transformants, based on results of ECR root staining, organic acid exudation and Al accumulation. In general, lines harboring the PPDK construct were more tolerant to Al stress than those expressing the ΔPPDK construct. We conclude that the overexpression of PPDK can serve to protect roots against Al toxicity. Furthermore, we provide the first physiological evidence for a link between Al stress and PPDK in plants and postulate that such approach could be successful under certain edaphic conditions, namely acid soils.

To demonstrate an asymmetry of the internal jugular veins, a finding which will have consequences for catheterization. Prospective study. The medical ICU of a university-affiliated teaching hospital. Eighty critically ill consecutive patients. Measurement of the cross-sectional area of the internal jugular veins. Search for an asymmetry, defined as an area at least twice that of the contralateral vein. An asymmetry was noted in 62.5% of the patients. The dominant vein was the right in only 68 % of these cases. In addition, 23% of the 160 jugular internal veins had an area of 0.4 cm2 or less. Using a simple technique, ultrasound identifies the dominant internal jugular vein, thus indicating the safer side before blind catheterization.

Immune cells become increasingly attractive as delivery system for immunotoxins in cancer therapy to reduce the intrinsic toxicity and severe side effects of chimeric protein toxins. In this study, we investigated the potential of human primary T cells to deliver a secreted immunotoxin through transient messenger RNA (mRNA) transfection. The chimeric protein toxin was directed toward the neovasculature of cancer cells by fusing a truncated version of Pseudomonas exotoxin A (PE38) to human vascular endothelial growth factor (VEGF) and to the single chain variable fragment (scFv) of anti-Her2/neu. Protocols for the transient transfection of human embryonic kidney cells (HEK293) as well as activated primary human T cells were established. Transient transfection with mRNA coding for the immunotoxins e23-PE38, VEGF-PE38 and its attenuated variant VEGF-PE38D yielded efficient expression and secretion. Mass spectrometry analysis endorsed that a fraction of VEGF-PE38D was properly translocated into the endoplasmic reticulum. Furthermore, cytotoxic activity of immunotoxin secreting T cells toward cancer cells was confirmed in co-culture with ovarian adenocarcinoma cells in the presence of a bispecific antibody (bsAb), highlighting the potential of primary T cells for mRNA-mediated immunotoxin delivery.

Fusion proteins for cell surface expression in the yeast Saccharomyces cerevisiae were constructed that consisted of the N-terminal leader sequence of Kre1p, followed by the nine amino acid viral epitope hemagglutinin (HA), and the carboxyterminal anchoring domain of either Cwp2p or Flo1p. All fusions were constitutively expressed under transcriptional control of the phosphoglycerate kinase promoter and immunofluorescence analysis indicated that in each construct the HA peptide was correctly anchored to the outer yeast cell surface. Successful solubilization of the cell wall fusions by laminarinase treatment indicated that the fusions are covalently linked to cell wall beta-1,3-D-glucans in vivo. FACS analyses further demonstrated that 70% of the yeast cell population expressed the corresponding cell wall fusion. Neither the number of positive cells within the population nor the distribution of the fusion at the single-cell level were negatively affected by replacing the \"heterologous\" Kre1p leader by the \"native\" Cwp2p leader. Insertion of a 350 amino acid Ser/Thr-rich spacer sequence into the fusions led to a dramatic increase in HA peptide accessibility on the yeast cell surface. Our data show that FACS analyses represent a valuable means for investigating cell surface expression, and indicate that artificial-spacer-elongated cell wall fusions might raise novel possibilities for cell surface expression of heterologous proteins in yeast.

Previous studies underlined the capacity of recombinant yeast as efficient vehicle for the targeted delivery of functional nucleic acids as well as proteinaceous antigens to mammalian antigen-presenting cells (APCs). To improve this yeast-mediated cargo transport into APCs, we investigated the impact of coexpression of the human membrane-perturbing protein perforin in comparison with bacterial listeriolysin O (LLO) on the yeast-based delivery of DNA, mRNA and proteins to mammalian APCs. In contrast to LLO, a cholesterol-dependent pore-forming toxin of Listeria , intracellular expression of human perforin in Saccharomyces cerevisiae had no impact on yeast cell viability, while its coexpression significantly increased translocation of ovalbumin and subsequent activation of ovalbumin-specific T lymphocytes. Likewise, perforin improved the expression of the model antigen enhanced green fluorescent protein after yeast-mediated DNA and mRNA delivery, whereas LLO was only able to enhance DNA delivery. Taken together, our data show that human perforin, besides bacterial hemolysins, represents a promising means to improve the yeast-mediated delivery of functional nucleic acids and proteins to mammalian APCs.

Activation of the NF-kappa B/Rel family of transcription factors is regulated by a cytoplasmic inhibitor, I kappa B alpha. Activity of I kappa B alpha is in turn modulated by phosphorylation and proteolysis. It has been postulated that phosphorylation of I kappa B alpha leads to its dissociation from NF-kappa B, and free I kappa B alpha is targeted for rapid degradation. However, this phosphorylation-mediated dissociation event has not been demonstrated in vivo. We demonstrate that, contrary to this hypothesis, phosphorylation of I kappa B alpha induced by tumor necrosis factor alpha in HeLa cells does not induce dissociation. We propose a model in which (i) induced phosphorylation of I kappa B alpha does not result in its dissociation from NF-kappa B, (ii) phosphorylation of I kappa B alpha serves as a signal for degradation, and (iii) degradation of I kappa B alpha occurs while it is still complexed with NF-kappa B.

We have used isolated spinach (Spinacea oleracea L.) thylakoid membranes to investigate the possible cryoprotective properties of class I beta-1,3-glucanase (1,3-beta-D-glucan 3-glucanohydrolase; EC 3.2.1.39) and chitinase. Class I beta-1,3-glucanase that was purified from tobacco (Nicotiana tabacum L.) protected thylakoids against freeze-thaw injury in our in vitro assays, whereas class I chitinase from tobacco had no effect under the same conditions. The beta-1,3-glucanase acted by reducing the influx of solutes into the membrane vesicles during freezing and thereby reduced osmotic stress and vesicle rupture during thawing. Western blots probed with antibodies directed against tobacco class I beta-1,3-glucanase showed that in spinach and cabbage (Brassica oleracea L.) leaves an isoform of 41 kD was accumulated during frost hardening under natural conditions

Direct-drive implosions of DT-filled plastic-shells have been conducted at the Omega laser facility, measuring nuclear yields while varying Knudsen numbers (i.e., the ratio of mean free path of fusing ions to the length of fuel region) by adjusting both shell thickness (e.g., 7.5, 15, 20, 30 μm) and fill pressure (e.g., 2, 5, 15 atm). The fusion reactivity reduction model showed a stronger effect on yield as the Knudsen number increases (or the shell thickness decreases). The Reduced-Ion-Kinetic (RIK) simulation which includes both fusion reactivity reduction and mix model was necessary to provide a better match between the observed neutron yields and those simulated.

The virally encoded K28 toxin of Saccharomyces cerevisiae kills sensitive yeast cells in a multi-step receptor-mediated fashion by cell cycle arrest and inhibition of DNA synthesis. In vivo, the toxin is translated as a 38 kDa preprotoxin (pptox) which is enzymatically processed to the biologically active alpha/beta heterodimer during passage through the yeast secretory pathway. Here, we demonstrate that Schizosaccharomyces pombe, a yeast from which no natural toxin-secreting killer strains are known, is perfectly capable of expressing a killer phenotype. Episomal as well as integrating K28 pptox gene cassettes were constructed that allowed a tightly thiamine-regulated killer phenotype expression under transcriptional control of the Sch. pombe nmt1 promotor. Northern analysis of the toxin-coding transcript as well as Western analysis of the secreted toxin indicated that fission yeast is capable of expressing a correctly processed and fully functional virus toxin. Moreover, toxin secretion in recombinant Sch. pombe was at least tenfold higher than in any natural and/or recombinant Sac. cerevisiae killer strain, indicating that pptox-derived vectors might be attractive in the fast growing field of heterologous protein expression and secretion in yeast.