The role of the plasma-membrane Ca2+-ATPase in ca2+ homeostasis in Sinapis alba root hairs

The regulation of cytosolic Ca2+ has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca2+-ATPase. For this purpose, erythrosin B was used to inhibit the Ca2+-ATPase, and the Ca2+ ionophore A23187 was applied to manipulate cytosolic free [Ca2+] which was then measured with Ca2+-selective microelectrodes. (i) At 0.01 M, A23187 had no effect on the membrane potential but enhanced the Ca2+ permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A23187 first caused a decrease in cytosolic Ca2+ by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca2+ spontaneously reversed and became an increase, a process which strongly depended on the external Ca2+ concentration, (iv) Upon removal of A23187, the cytosolic free [Ca2+] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca2+ is an activation of Ca2+ transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca2+ from the cytosol. The two observations that after the addition of A23187, (i) Ca2+ gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca2+-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH+/Ca2+-ATPase. Based on the finding that the Ca2+-ATPase inhibitor erythrosin B had no effect on cytosolic Ca2+, but caused a strong Ca2+ increase after the addion of A23187 we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca2+-pump. We further conclude that this ATPase is a major Ca2+ regulator in stress situations where the cytosolic Ca2+ has been shifted from its steady-state level, as may be the case during processes of signal transduction

Signal Transduction in Sinapis alba Root Hairs: Auxins as External Messengers

In developing root hairs of Sinapis alba the effects of externally applied indole-3-acetic acid (IAA) and other auxins have been investigated with respect to membrane potential, membrane conductance, cytosolic Ca2 + and pH. Following a delay of roughly 30s, 10- 12 to lO- IO M IAA slowly hyperpolarize, 10- 7 M IAA rapidly depolarize the root hairs, while 10- 9 M has hardly an effect. We show that these voltage responses are not the result of a change in membrane conductance or permeability, but are presumably caused by a change in H+ ATPase activity. The other tested auxins and analogues yielded comparable effects, but with much lower effectivity (IAA > 1-NAA > 2,4-D ~ 2-NAA > 2,3-D). Cytosolic Ca2+ and pH were decreased during depolarization by 0.2 and 0.4 units, respectively. No such changes were observed during hyperpolarization or about 1 h after the first encounter of the root hairs with IAA. We propose that IAA is a natural external signal for roots while competing with neighboring organisms for nutrients and salts, and suggest a signal chain with the plasma membrane H+ ATPase as a target protein. The delay in response to IAA, the time dependency, and the extremely low effective IAA concentrations point to the existence of a IAA receptor. Since the IAA-induced shifts in cytosolic pH and Ca2+ occur simultaneously with the depolarization, the question whether these ions are cellular messengers and part of an IAA-triggered signal chain is critically discussed

CD154 Expression Indicates T Cell Activation Following Tetanus Toxoid Vaccination of Horses.

Despite the relevance of adaptive immunity against equine pathogens antigen-specific T cell responses of horses are not well characterized and the lack of insight into T cell responses hampers the understanding of the pathogeneses of important diseases. In this study we used tetanus toxoid (TT) as a well-defined antigen to characterize antigen-reactive T cells. Six healthy adult horses received a routine booster against tetanus with an immune stimulating complex (ISCOM)-based vaccine and were followed for 28 days. TT-specific serum antibodies were quantified by ELISA and increased in all horses by day 7 after vaccination. CD154 is an established indicator of antigen-reactive T helper cells in other species, but has not been characterized in horses. CD154 detection in equine PBMC by an anti-human CD154 antibody (clone 5C8) was confirmed by Western blots and then applied for flow cytometry. As a common indicator of equine T cell activation, cytokine induction was studied in parallel. T cells were analyzed by multicolor flow cytometry of PBMC after re-stimulation with TT in vitro. Reactive T helper (Th) cells were characterized by increased frequencies of CD4+CD154+ lymphocytes in in vitro TT-re-stimulated PBMC on day 14 after vaccination of the horses compared to pre-vaccination. The majority of all CD154+ cells after TT re-stimulation were CD4+ Th cells, but CD154 was also induced on CD4- cells albeit in lower frequencies. CD154+CD4+ Th cells were enriched in cytokine-expressing cells compared to CD154-CD4+ Th cells. Similar to the CD4+CD154+ frequencies, CD4+IL-4+, CD4+IFN-γ+ and CD4+TNF-α+ were increased after vaccination, but IL-4+ increased later than IFN-γ+ and CD4+TNF-α+, which already exceeded pre-vaccination frequencies on day 7. CD4+CD154+ frequencies correlated positively with those of CD4+IL-4+ (Th2) on day 14, and negatively with CD4+IFN-γ+ induction on day 7, but did not correlate with CD4+TNF-α+ frequencies or TT-specific antibody concentrations. CD154 appears to be a useful marker of antigen-reactive equine Th cells in combination with cytokine expression. The T cell analyses established here with TT can be applied to other antigens relevant for infections or allergies of horses and in horse models for translational research

Trimethylsilyl tag for probing protein-ligand interactions by NMR

Protein-ligand titrations can readily be monitored with a trimethylsilyl (TMS) tag. Owing to the intensity, narrow line shape and unique chemical shift of a TMS group, dissociation constants can be determined from straightforward 1D 1H-NMR spectra not only in the fast but also in the slow exchange limit. The tag is easily attached to cysteine residues and a sensitive reporter of ligand binding also at sites where it does not interfere with ligand binding or catalytic efficiency of the target protein. Its utility is demonstrated for the Zika virus NS2B-NS3 protease and the human prolyl isomerase FK506 binding protein.C.N. and G.O. thank the Alexander von Humboldt Foundation for a Feodor Lynen Fellowship and the Australian Research Council for a Laureate Fellowship, respectively. Financial project support by the Australian Research Council, the Austrian Science Fund (FWF) (DK Molecular Enzymology W901 to K.Z.) and by NAWI Graz is gratefully acknowledged

Color Change Effect in an Organic-Inorganic Hybrid Material Based on a Porphyrin Diacid

Porphyrinic materials show a range of interesting and useful optical and electrical properties. The less well-known sub-class of porphyrin diacids has been used in this work to construct an ionic hybrid organic-inorganic material in combination with a halogenidometalate anion. The resulting compound, $[H_6TPyP][BiCl_6]_2$ (1) (TPyP = tetra(4-pyridyl)porphyrin) has been obtained via a facile solution based synthesis in single crystalline form. The material exhibits a broad photoluminescence emission band between 650 and 850 nm at room temperature. Single crystals of $[H_6TPyP][BiCl_6]_2$ show a photocurrent in the fA and a much higher dark current in the nA range. They also display an unexpected reversible color change upon wetting with different liquids. This phenomenon has been investigated with optical spectroscopy, SEM, XPS and NEXAFS techniques, showing that a surface-based structural coloration effect is the source of the color change. This stands in contrast to other materials where structural coloration typically has to be introduced through elaborate, multi-step processes or the use of natural templates. Additionally, it underscores the potential of self-assembly of porphyrinic hybrid compounds in the fabrication of materials with unusual optical properties

Reading Self-Efficacy Predicts Word Reading But Not Comprehension in Both Girls and Boys

The relationship between cognitive skills and reading has been well-established. However, the role of motivational factors such as self-efficacy in reading progress is less clear. In particular, it is not clear how self-efficacy relates to word level reading versus comprehension, and whether this differs in boys and girls. This study examines the relationship between self-efficacy, word reading and reading comprehension across the range of reading abilities after controlling for reading-related cognitive factors. One hundred and seventy nine children (86 males and 93 females) between 8 and 11 years old completed a self-report measure of reading self-efficacy together with measures of reading comprehension and word reading, working memory, auditory short-term memory, phonological awareness, and vocabulary. Boys and girls showed similar levels of attainment and reading self-efficacy. Reading self-efficacy was associated with word reading, but not with reading comprehension in either boys or girls. It is argued that this may reflect important differences between reading self-efficacy and more general measures of reading motivation and engagement. Reading self-efficacy is an element of reading motivation that is closely associated with a child’s perceived attainments in reading and is less susceptible to the gender differences seen in broader measures

Polarisation of the omega meson in the pd-->3He+omega reaction at 1360 and 1450 MeV

The tensor polarisation of omega mesons produced in the pd-->3He+omega reaction has been studied at two energies near threshold. The 3He nuclei were detected in coincidence with the pi0pi+pi- or pi0gamma decay products of the omega. In contrast to the case of phi meson production, the omega mesons are found to be unpolarised. This brings into question the applicability of the Okubo-Zweig-Iizuka rule when comparing the production of vector mesons in low energy hadronic reactions.Comment: 11 pages, 4 figure

Systematic study of the pp -> pp omega reaction

A systematic study of the production of omega-mesons in proton-proton-collisions was carried out in a kinematically complete experiment at three excess energies(epsilon= 92, 128, 173MeV). Both protons were detected using the large-acceptance COSY-TOF spectrometer at an external beam line at the Cooler Synchrotron COSY at Forschungszentrum J\"ulich. The total cross section, angular distributions of both omega-mesons and protons were measured and presented in various reference frames such as the overall CMS, helicity and Jackson frame. In addition, the orientation of the omega-spin and invariant mass spectra were determined. We observe omega-production to take place dominantly in Ss and Sp final states at epsilon = 92, 128 MeV and, additionally, in Sd at epsilon= 173 MeV. No obvious indication of resonant omega-production via N^*-resonances was found, as proton angular distributions are almost isotropic and invariant mass spectra are compatible with phase space distributions. A dominant role of ^3P_1 and ^1S_0 initial partial waves for omega-production was concluded from the orientation of the decay plane of the omega-meson. Although the Jackson angle distributions in the omega-p-Jackson frame are anisotropic we argue that this is not an indication of a resonance but rather a kinematical effect reflecting the anisotropy of the omega angular distribution. The helicity angle distribution in the omega-p-helicity frame shows an anisotropy which probably reflects effects of the omega angular momenta in the final state; this observable may be, in addition to the orientation of the omega decay plane, the most sensitive one to judge the validity of theoretical descriptions of the production process.Comment: 17 pages, 16 figures, accepted for publication in EPJ