13 research outputs found

    Streptococcus pneumoniae Serotype 1 Capsular Polysaccharide Induces CD8+CD28− Regulatory T Lymphocytes by TCR Crosslinking

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    Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an α-helix configuration. In this paper we look at the immune response of CD8+ T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8+CD28− T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8+CD28− T lymphocytes. The Sp1-induced CD8+CD28− T lymphocytes are CD122lowCTLA-4+CD39+. They synthesize IL-10 and TGF-β. The Sp1-induced CD8+CD28− T cells exhibit immunosuppressive properties on CD4+ T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8+ T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8+ T cell activation by enhancing crosslinking of TCR. The expansion of CD8+CD28− T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8+CD28− T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-κB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8+CD28− population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8+CD28− T lymphocytes in vivo and in vitro. The underlying mechanism of CD8+ T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system

    Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants

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    Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (−130 mV to −110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. −100 mV). In roots, the membrane potential of sieve elements dropped abruptly to −55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H+-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie–Hofstee (EH) -transformations pointed at biphasic Michaelis–Menten kinetics (2 MM, EH: Km1 1.2–1.8 mM, Km2 6.6–9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, Km values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher Km values (EH: Km1 10 mM, Km2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (−0.1 to −0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) Km values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of Km values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose uptake by sieve elements, (d) Affinity for sucrose uptake by sieve elements exceeds by far that by phloem parenchyma cells, (e) Patch-clamp studies provide a feasible basis for quantification of sucrose uptake by single cells. The consequences of the findings for whole-plant carbohydrate partitioning are discussed.Peer Reviewe

    Functional Sieve Element Protoplasts1[OA]

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    Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts—two protoplasts intermitted by a sieve plate—of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling


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    Sieve Element Ca2+ Channels as Relay Stations between Remote Stimuli and Sieve Tube Occlusion in Vicia faba[W]

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    Damage induces remote occlusion of sieve tubes in Vicia faba by forisome dispersion, triggered during the passage of an electropotential wave (EPW). This study addresses the role of Ca2+ channels and cytosolic Ca2+ elevation as a link between EPWs and forisome dispersion. Ca2+ channel antagonists affect the initial phase of the EPW as well as the prolonged plateau phase. Resting levels of sieve tube Ca2+ of ∼50 nM were independently estimated using Ca2+-selective electrodes and a Ca2+-sensitive dye. Transient changes in cytosolic Ca2+ were observed in phloem tissue in response to remote stimuli and showed profiles similar to those of EPWs. The measured elevation of Ca2+ in sieve tubes was below the threshold necessary for forisome dispersion. Therefore, forisomes need to be associated with Ca2+ release sites. We found an association between forisomes and endoplasmic reticulum (ER) at sieve plates and pore-plasmodesma units where high-affinity binding of a fluorescent Ca2+ channel blocker mapped an increased density of Ca2+ channels. In conclusion, propagation of EPWs in response to remote stimuli is linked to forisome dispersion through transiently high levels of parietal Ca2+, release of which depends on both plasma membrane and ER Ca2+ channels