Shaping of adaptive immune responses to soluble proteins by TLR agonists: a role for IFN-alpha/beta

Toll-like receptors (TLR) are believed to play a major role in the recognition of invading organisms, although their ability to shape immune responses is not completely understood. Our aim was to investigate in vivo the effect of different TLR stimuli on the generation of antibody responses and the induction of CD8+ T-cell cross-priming after immunization with soluble protein antigens. While all TLR agonists tested elicited the production of immunomodulatory cytokines, marked differences were observed in their ability to stimulate antigen-specific immune responses. Zymosan, poly(I:C) and CpG DNA, which signal through TLR2/6, 3 and 9, respectively, were found to strongly induce the production of IgG2a antibodies, whereas R-848 (TLR7) and LPS (TLR4) did so much more weakly. In contrast, LPS, poly(I:C) and CpG DNA, but not zymosan, induced functional CD8+ T-cell responses against OVA; peptidoglycan (TLR2/?) and R-848 were also ineffective in stimulating cross-priming. Experiments using IFN-alpha/beta R-deficient mice showed that the induction of cross-priming by LPS and poly(I:C) was abrogated in the absence of IFN-alpha/beta signalling, and induction by CpG DNA was greatly reduced. Overall, our results identify LPS as another TLR agonist that is able to generate functional cross-priming against a soluble protein antigen. In addition, our results demonstrate that the ability of TLR stimuli to initiate CD8+ T-cell responses against soluble protein antigens is largely dependent on the IFN-alpha/beta signalling pathway.     

Bcl-2 and tBid proteins counter-regulate mitochondrial potassium transport

The mechanism of cytochrome c release from mitochondria in apoptosis remains obscure, although it is known to be regulated by bcl-2 family proteins. Here we describe a set of novel apoptotic phenomena—stimulation of the mitochondrial potassium uptake preceding cytochrome c release and regulation of such potassium uptake by bcl-2 family proteins. As a result of increased potassium uptake, mitochondria undergo moderate swelling sufficient to release cytochrome c. Overexpression of bcl-2 protein prevented the mitochondrial potassium uptake as well as cytochrome c release in apoptosis. Bcl-2 was found to upregulate the mitochondrial potassium efflux mechanism—the K/H exchanger. Specific activation of the mitochondrial K-uniporter led to cytochrome c release, which was inhibited by bcl-2. tBid had an opposite effect—it stimulated mitochondrial potassium uptake resulting in cytochrome c release. The described counter-regulation of mitochondrial potassium transport by bcl-2 and Bid suggests a novel view of a mechanism of cytochrome c release from mitochondria in apoptosis.     

Plant MCM proteins: role in DNA replication and beyond

Mini-chromosome maintenance (MCM) proteins form heterohexameric complex (MCM2–7) to serve as licensing factor for DNA replication to make sure that genomic DNA is replicated completely and accurately once during S phase in a single cell cycle. MCMs were initially identified in yeast for their role in plasmid replication or cell cycle progression. Each of six MCM contains highly conserved sequence called “MCM box”, which contains two ATPase consensus Walker A and Walker B motifs. Studies on MCM proteins showed that (a) the replication origins are licensed by stable binding of MCM2–7 to form pre-RC (pre-replicative complex) during G1 phase of the cell cycle, (b) the activation of MCM proteins by CDKs (cyclin-dependent kinases) and DDKs (Dbf4-dependent kinases) and their helicase activity are important for pre-RC to initiate the DNA replication, and (c) the release of MCMs from chromatin renders the origins “unlicensed”. DNA replication licensing in plant is, in general, less characterized. The MCMs have been reported from Arabidopsis, maize, tobacco, pea and rice, where they are found to be highly expressed in dividing tissues such as shoot apex and root tips, localized in nucleus and cytosol and play important role in DNA replication, megagametophyte and embryo development. The identification of six MCM coding genes from pea and Arabidopsis suggest six distinct classes of MCM protein in higher plant, and the conserved function right across the eukaryotes. This overview of MCMs contains an emphasis on MCMs from plants and the novel role of MCM6 in abiotic stress tolerance.     

Sucrose regulates plant responses to deficiencies in multiple nutrients

Arabidopsis mutant hps1 that over-accumulates sucrose has enhanced sensitivity in almost all the aspects of plant responses to phosphate starvation. The detailed characterization of hps1 has led to the conclusion that sucrose is a global regulator of plant phosphate responses. Here, we show that hps1 is also hypersensitive to nitrogen and potassium deprivation, as well as to decreased levels of overall macronutrients. These results suggest that sucrose regulates plant deficiency responses to multiple nutrients and is part of a general response to nutrient deprivation.Key words: nutrient deficiency, plant response, hps1, sucrose, signalingPlants often encounter nutrient deficiency in their surrounding environments,¹ and because they are sessile, plants have to develop sophisticated strategies to cope with nutritional stress. The strategies include an array of biochemical, physiological, and developmental responses.^2–5 Interactions among the different nutrients have also been documented.^6,7 For example, ammonium interacts with potassium (K) for uptake,⁸ and nitrate transporters were downregulated in K-deprived plants.⁹ Phosphate (P) deficiency induces expression of the K transporter gene HAK5 and causes high accumulation of iron.^7,10,11 The intimate cross talk among the different nutrients suggests the existence of some common signaling components that are involved in regulating plant responses to different nutrient stresses. However, the molecular identity of these signaling components remains unknown.Previously, we reported the characterization of an Arabidopsis mutant hps1.¹⁰ In hps1, a sucrose transporter gene, SUC2, is overexpressed, resulting in enhanced uptake of sucrose from the culture medium. As a consequence, hps1 accumulates a high level of sucrose in both its shoot and root tissues. hps1 is hypersensitive in almost all the aspects of plant responses to P starvation. In contrast, the suc2 knockout mutant displays opposite phenotypes. We also showed that a high level of sucrose in hps1 can directly induce most P starvation-responsive genes even under P sufficient condition. Under normal growth condition, hps1 contains less chlorophyll, probably due to the feedback inhibition of photosynthesis by the high level of sucrose. Combined with the results from our genomic research and early work by other groups,^10,12 we conclude that sucrose is a global regulator of plant responses to P starvation. In this study we determine if sucrose is also involved in responses to other nutrients and thus is part of a general response mechanism to nutrient deprivation.We first tested whether hps1 also shows hypersensitivity to nitrogen (N) and K deprivation. The seeds of the WT and hps1 were directly sown on MS medium in which sucrose was replaced by glucose to ensure the same growth status. Five days after seed germination, the seedling were transferred to sucrose-containing MS medium without supplementation of N, P or K, respectively, and grow for another 5 or 12 days. Compared to the WT, P-starved hps1 had smaller size of shoots and accumulated more anthocyanin (Fig. 1A and B); this was consistent with our previous findings.¹⁰ Similarly, hps1 showed enhanced sensitivity to N and K deprivation in term of primary root growth (Fig. 1A). N starved-hps1 plants also accumulated more anthocyanins than N starved-WT plants (Fig. 1B). However, there was no significant difference of the shoot size between the WT and hps1 on N and K deficiency medium.Open in a separate windowFigure 1The hps1 mutant is hypersensitive to N, P and K deprivation. Five-day-old seedlings of the WT and hps1 mutant grown on glucose-containing MS medium were transferred to sucrose-containing MS medium with or without supplemented phosphate, nitrogen or potassium. The seedlings were photographed 5 days (A) and 12 days (B) after transfer. Bar = 10 mm.To further determine whether hps1 is hypersensitive to a general decrease in nutrient supply, we examined the responses of hps1 grown on sucrose-containing MS medium with different levels of nutrients. When grown on full-strength MS medium, shoot size and shoot fresh weight were similar for the WT and hps1 (Fig. 2). Decreasing the macronutrient concentration to 1/2 strength significantly enhanced the shoot growth of both the WT and hps1. Reducing the macro-nutrient concentration to 1/10 had no further obvious effect on the WT, but it dramatically inhibited the shoot growth of hps1 (Fig. 2A and B). Ten days after seed germination, the shoot fresh weight of hps1 grown on 1/20 MS medium was only about 15% of that for the WT. Regardless of the strength of the MS media, no accumulation of anthocyanin could be visually detected for the WT. For the hps1 mutant, however, anthocyanin accumulation was evident even on 1/4 MS medium, and the accumulation increased as the macronutrient level was further decreased (Fig. 2A). Interestingly, the hypersensitive responses of hps1 to decreased macronutrient concentration only occurred in shoot growth, but not in root growth (Fig. 2A and C).Open in a separate windowFigure 2The hps1 mutant is hypersensitive to a decreased level of macronutrients. Five-day-old seedlings of the WT and hps1 mutant grown on glucose-containing MS medium were transferred to sucrose-containing MS medium with different strength of macronutrients. After transfer, the seedlings were grown for another 10 days. (A) Photographs of the WT and hps1 seedlings after growth on the MS medium with different nutrient strength. (B and C): Shoot and root fresh weight (FW) of the WT and hps1 seedlings shown in (A). Data represents means ± SE (n > 10). Values with different letters differ significantly (p < 0.01). Bar = 10 mm.     

The role of SMC proteins in the responses to DNA damage

The SMC proteins form the cores of three protein complexes in eukaryotes, cohesin, condensin and the Smc5-6 complex. Cohesin holds sister chromatids together after DNA replication and is involved in both the repair of double-strand breaks by homologous recombination and the intra-S-phase checkpoint. Condensin assists in the condensation of chromosomes at mitosis and also has a role in checkpoint control pathways. The Smc5-6 complex is involved in a variety of DNA repair and damage response pathways by as yet unknown mechanisms, but is also associated with repair by homologous recombination.     

Guard cell responses to potassium ferricyanide

Micromolar concentrations of potassium ferricyanide inhibit light-induced stomatal opening. The extent of the inhibition is dependent on the presence of carbon dioxide and the concentration of potassium ferricyanide needed to obtain 50% inhibition of stomatal opening is 40-fold higher in CO₂-free air than in normal air. The fungal toxin, fusicoccin (1 μ M ), overcame the ferricyanide inhibition of stomatal opening indicating that the electron acceptor may interact more or less directly with the activity of the plasma membrane H⁺-ATPase. Although potassium ferricyanide strongly inhibited stomatal opening, it had only minor effects on stomatal maintaining or stomatal closure due to darkness or ABA.     

The role of sodium and potassium transport pathways in taste transduction: an overview

Recent studies have shown that taste sensations are mediatedby a multiplicity of transduction mechanisms. The taste of saltis produced in part by the entry of Na⁺ through channels inthe apical taste cell membrane. Na⁺ transport also mediatessweet perception in some species. The taste of KCI requiresentry of K⁺ through apical potassium channels. The productionof second messengers such as cAMP by taste stimuli or tastemodifiers can depolarize taste cells by inducing an enzymaticcascade that alters K⁺ permeability.