Sensing and refilling calcium stores in an excitable cell.

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.     

Interactions between elicitins and radish Raphanus sativus

Two responses to elicitins are described in cultivars of radish (Raphanus sativus L.). Type I, exhibited by the cultivar Daikon, is characterised by wilting and desiccation within 24 h of elicitin application and was previously reported as the sensitive response (S. Kamoun et al. 1993, Mol Plant-Microbe Interact 6: 15–25). At 1 μg elicitin · g⁻¹ FW radish tissue, symptoms appeared after 8 h, a sensitivity comparable to that shown by tobacco to β elicitins (J.-C. Pernollet et al., 1993, Physiol Mol Plant Pathol 42: 53–67; S. Kamoun et al., 1993, Mol Plant-Microbe Interact 6: 15–25). Elicitin failed to induce these symptoms in the cultivar White Icicle, even at 100 μg · g⁻¹ FW of tissue. However, a different response (Type II) with symptoms resembling senescence appeared in White Icicle after 48 h and were fully developed by 72 h. The Type II response was induced at levels of elicitin above 0.3 μg · g⁻¹ FW. Elicitin-treated Daikon leaves held at 100% relative humidity, rather than ambient (50–60%) did not wilt and by 72 h displayed Type II symptoms. When treated Daikon leaves were removed to ambient humidity at any time during the latent period, they developed Type I symptoms within 2 h. Although Type I symptoms were suppressed in Daikon at high humidity, there was no indication that leaf diffusion resistance or plant water conductance were affected. Protoplasts from the cultivar Daikon responded to elicitin by H⁺ uptake and K⁺ release, with maximal response at 300 pM. The response was eliminated by K252a or staurosporine. Daikon protoplasts also showed transient uptake/secretion of Ca²⁺ on elicitin addition. Protoplasts from White Icicle gave neither of these responses. Both Daikon and White Icicle phenotypes could be transferred to progeny of Daikon-White Icicle crosses and in the F2 generation three phenotypes, including a null, segregated. Only those F2 plants which exhibited the Daikon phenotype produced protoplasts which responded to elicitin. Received: 13 May 1997 / Accepted: 27 August 1997     

The effect of n-propyl gallate on the formation of ethylene during protoplast isolation in sugarbeet (Beta vulgaris L.)

The formation of ethylene during isolation of mesophyll protoplastsfrom leaves of in vitro cultured sugarbeet (Beta vulgaris L.)seedlings was monitored. The addition of the lipoxygenase (EC1.13.11.12 [EC] ) inhibitor n-propyl gallate to the isolation mediumsignificantly reduced the amount and rate of ethylene production.The relevance of cell physiology on protoplast regenerationis discussed. Key words: Beta vulgaris L., protoplasts, ethylene, lipoxygenase inhibitor, regeneration     

Transport of trehalose in Salmonella typhimurium.

We have studied trehalose uptake in Salmonella typhimurium and the possible involvement of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in this process. Two transport systems could recognize and transport trehalose, the mannose PTS and the galactose permease. Uptake of trehalose via the latter system required that it be expressed constitutively (due to a galR or galC mutation). Introduction of a ptsM mutation, resulting in a defective IIMan/IIIMan system, in S. typhimurium strains that grew on trehalose abolished growth on trehalose. A ptsG mutation, eliminating IIGlc of the glucose PTS, had no effect. In contrast, a crr mutation that resulted in the absence of IIIGlc of the glucose PTS prevented growth on trehalose. The inability of crr and also cya mutants to grow on trehalose was due to lowered intracellular cyclic AMP synthesis, since addition of extracellular cyclic AMP restored growth. Subsequent trehalose metabolism could be via a trehalose phosphate hydrolase, if trehalose phosphate was formed via the PTS, or trehalase. Trehalose-grown cells contained trehalase activity, but we could not detect phosphoenolpyruvate-dependent phosphorylation of trehalose in toluenized cells.     

The BECN1 N-terminal domain is intrinsically disordered

BECN1/Beclin 1 has a critical role in the early stages of autophagosome formation. Recently, structures of its central and C-terminal domains were reported, however, little structural information is available on the N-terminal domain, comprising a third of the protein. This lack of structural information largely stems from the inability to produce this region in a purified form. Here, we describe the expression and purification of the N-terminal domain of BECN1 (residues 1 to 150) and detailed biophysical characterization, including NMR spectroscopy. Combined, our studies demonstrated at the atomic level that the BECN1 N-terminal domain is intrinsically disordered, and apart from the BH3 subdomain, remains disordered following interaction with a binding partner, BCL2L1/BCL-X_L. In addition, the BH3 domain α-helix induced upon interaction with BCL2L1 reverts to a disordered state when the complex is dissociated by exposure to a competitive inhibitor. No significant interactions between N- and C-terminal domains were detected.     

Recombinant HLA-DP2 binds beryllium and tolerizes beryllium-specific pathogenic CD4+ T cells

Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the alpha-1 and beta-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4 -pulsed, plate-bound rHLA-DP2 molecules induced IFN-gamma secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag.     

The Importance of Demonstratively Restoring Order

Contrary to what is often assumed, order is not the strongest context for encouraging normative behavior. The strongest context effect on normative behavior comes from cues that clearly convey other people’s respect for norms. Ironically, this show of respect necessitates some contrasting disrespect that is being restored. Using civic virtues (such as helping behavior) as a prototype of normative behavior, the three field experiments described in this paper reveal the impact of normative cues on civic virtues. Results show that the strongest effect on making people follow prosocial norms in public places emanates from seeing order being restored, rather than just order being present. The robust and surprisingly large effects show that observing other people’s respect for one particular norm (as evidenced in their restoring physical order) makes it more likely that the onlooker follows other norms as well. This implies that prosocial behavior has the highest chance of spreading when people observe order being restored. There are clear policy implications: create low cost “normative respect cues” wherever it is desirable to increase conformity to norms.