Leishmania amazonensis: Anionic currents expressed in oocytes upon microinjection of mRNA from the parasite

Transport mechanisms involved in pH homeostasis are relevant for the survival of Leishmania parasites. The presence of chloride conductive pathways in Leishmania has been anticipated since anion channel inhibitors limit the proton extrusion mediated by the H+ATPase, which is the major regulator of intracellular pH in amastigotes. In this study, we used Xenopus laevis oocytes as a heterologous expression system in which to study the expression of ion channels upon microinjection of polyA mRNA from Leishmania amazonensis. After injection of polyA mRNA into the oocytes, we measured three different types of currents. We discuss the possible origin of each, and propose that Type 3 currents could be the result of the heterologous expression of proteins from Leishmania since they show different pharmacological and biophysical properties as compared to endogenous oocyte currents.     

Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K⁺ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (I_K1) and the carbacholine-induced inward rectifier (I_KACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal I_K1 of ventricular myocytes was blocked with apparent IC50-values of 4.6 × 10^− 6 M and 3.5 × 10^− 6 M for rat and fish, respectively. I_KACh was almost an order of magnitude more sensitive to KB-R7943 than I_K1 with IC₅₀-values of 6.2 × 10^− 7 M for rat and 2.5 × 10^− 7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3 × 10^− 6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order I_K1 ~ I_NCX < I_KACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular I_KACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.     

Inhibition of the calcium-activated chloride current in cardiac ventricular myocytes by N-(p-amylcinnamoyl)anthranilic acid (ACA)

N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A₂ (PLA₂) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca²⁺-activated chloride current (I_Cl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited I_Cl(Ca) in a concentration-dependent manner (IC₅₀ = 4.2 μM, n_Hill = 1.1), without affecting the L-type Ca²⁺ current. Unlike ACA, the non-selective PLA₂ inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on I_Cl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on I_Cl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides I_Cl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on I_Cl(Ca) are PLA₂-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.     

Leishmania amazonensis: xylitol as inhibitor of macrophage infection and stimulator of macrophage nitric oxide production

Xylitol is a sugar alcohol being explored for clinical uses. The aim was to evaluate the effects of xylitol on Leishmania amazonensis-infected J774A.1 macrophages. Macrophages were infected with L. amazonensis for 3h, washed and incubated with 2.5 or 5.0% xylitol for 24, 48, and 72 h at 37 degrees C. Infection indexes for macrophages incubated only in medium were compared to those treated with xylitol. Cell viability and nitric oxide production were determined each time. Xylitol did not affect L. amazonensis or J774A.1 cell viabilities. Xylitol at 5.0% stimulated nitric oxide production by macrophages at 72 h (p<0.01). At 2.5 and 5.0%, xylitol inhibited nitric oxide production by L. amazonensis at 48 h (p<0.05) when compared to control. Infection indexes were significantly lower at 72 h (p<0.05), (16.9% and 9.6%) in cells cultivated with 2.5 and 5.0% xylitol, respectively, compared to control (38.4%). Results suggest a potential leishmanicidal action of the xylitol on infected macrophages.     

Effects of protons on macroscopic and single-channel currents mediated by the human P2X7 receptor

Human P2X7 receptors (hP2X7Rs) belong to the P2X family, which opens an intrinsic cation channel when challenged by extracellular ATP. hP2X7Rs are expressed in cells of the inflammatory and immune system. During inflammation, ATP and protons are secreted into the interstitial fluid. Therefore, we investigated the effect of protons on the activation of hP2X7Rs. hP2X7Rs were expressed in Xenopus laevis oocytes and activated by the agonists ATP or benzoyl-benzoyl-ATP (BzATP) at different pH values. The protons reduced the hP2X7R-dependent cation current amplitude and slowed the current deactivation depending on the type and concentration of the agonist used. These effects can be explained by (i) the protonation of ATP, which reduces the effective concentration of the agonist ATP⁴⁻ at the high- and low-affinity ATP activation site of the hP2XR, and (ii) direct allosteric inhibition of the hP2X7R channel opening that follows ATP⁴⁻ binding to the low-affinity activation site. Due to the hampered activation via the low-affinity activation site, a low pH (as observed in inflamed tissues) leads to a relative increase in the contribution of the high-affinity activation site for hP2X7R channel opening.     

Mitochondrial chloride channels - What are they for?

This minireview focuses on observation of the properties, functional significance, and modulation of single chloride channels in the mitochondrial inner membrane using two electrophysiological methods - the patch-clamp and bilayer lipid membrane methods. Measurements of parameters such as conductance, Cl⁻/K⁺ selectivity, voltage or pH dependence as well as their modulation by endogenous and exogenous compounds using individual mitochondrial chloride channels result in an unexpectedly wide range of values. This paper discusses the origin of this wide variety of channel parameters and the possible involvement of these channels in mitochondrial membrane potential oscillations, apoptosis, carrier function, and mitochondrial fusion and fission.     

Taenia crassiceps: chloride currents expressed in Xenopus oocytes upon injection of mRNA of cysticerci (WFU strain) isolated from mice

To study the properties of ion channels of the tapeworm Taenia crassiceps, mRNA was isolated from cysticerci and injected into mature oocytes of the frog Xenopus laevis and ion currents were recorded four days after injection with the two-electrode voltage clamp technique. Oocytes injected with mRNA of T. crassiceps expressed outward currents (I_TC) that activated instantly after onset of the test pulse, followed by a slow inactivation at potentials over +40 mV, with a reversal potential of −23.2 ± 5 mV. They were not affected by changes on monovalent cationic composition of external media, but replacement of external chloride by gluconate shifted significantly the reversal potential, suggesting that I_TC are anion currents, with a permeability sequence of . These currents were sensitive to changes of external pH but not to hypotonic challenges. They were significantly inhibited by DIDS, NPPB and Niflumic acid, but not by 9-anthracene. These results suggest that I_TC are the result of expression of anion channels from the tapeworm T. crassiceps.     

On the advantages of using green light to study fluorescence yield changes in leaves

In photosynthetic chains, the kinetics of fluorescence yield depends on the photochemical rates at the level of both Photosystem I and II and thus on the absorption cross section of the photosynthetic units as well as on the coupling between light harvesting complexes and photosynthetic traps. A new set-up is described which, at variance with the commonly used set-ups, uses of a weakly absorbed light source (light-emitting diodes with maximum output at 520 nm) to excite the photosynthetic electron chain and probe the resulting fluorescence yield changes and their time course. This approach optimizes the homogeneity of the exciting light throughout the leaf and we show that this homogeneity narrows the distribution of the photochemical rates. Although the exciting light is weakly absorbed, the possibility to tune the intensity of the light emitting diodes allows one to reach photochemical rates ranging from 10⁴ s^− 1 to 0.25 s^− 1 rendering experimentally accessible different functional regimes. The variations of the fluorescence yield induced by the photosynthetic activity are qualitatively and quantitatively discussed. When illuminating dark-adapted leaves by a weak light, the kinetics of fluorescence changes displays a pronounced plateau which precedes the fluorescence increase reflecting the full reduction of the plastoquinone pool. We ascribe this plateau to the time delay needed to reduce the photosystem I electron acceptors.     

Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: The qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side

The dark recovery kinetics of the Chl a fluorescence transient (OJIP) after 15 min light adaptation were studied and interpreted with the help of simultaneously measured 820 nm transmission. The kinetics of the changes in the shape of the OJIP transient were related to the kinetics of the qE and qT components of non-photochemical quenching. The dark-relaxation of the qE coincided with a general increase of the fluorescence yield. Light adaptation caused the disappearance of the IP-phase (20-200 ms) of the OJIP-transient. The qT correlated with the recovery of the IP-phase and with a recovery of the re-reduction of P700⁺ and oxidized plastocyanin in the 20-200 ms time-range as derived from 820 nm transmission measurements. On the basis of these observations, the qT is interpreted to represent the inactivation kinetics of ferredoxin-NADP⁺-reductase (FNR). The activation state of FNR affects the fluorescence yield via its effect on the electron flow. The qT therefore represents a form of photochemical quenching. Increasing the light intensity of the probe pulse from 1800 to 15000 μmol photons m⁻² s⁻¹ did not qualitatively change the results. The presented observations imply that in light-adapted leaves, it is not possible to ‘close’ all reaction centers with a strong light pulse. This supports the hypothesis that in addition to Q_A a second modulator of the fluorescence yield located on the acceptor side of photosystem II (e.g., the occupancy of the Q_B-site) is needed to explain these results. Besides, some of our results indicate that in pea leaves state 2 to 1 transitions may contribute to the qI-phase.     

Subunit a of the F1F0 ATP Synthase Requires YidC and SecYEG for Membrane Insertion

The insertion of inner membrane proteins in Escherichia coli occurs almost exclusively via the SecYEG pathway, while some membrane proteins require the membrane protein insertase YidC. In vitro analysis demonstrates that subunit a of the F₁F₀ ATP synthase (F₀a) is strictly dependent on Ffh, SecYEG and YidC for its membrane insertion but independent of the proton motive force. The insertion of the first transmembrane segment of F₀a also depends on Ffh and SecYEG but not on YidC, whereas the insertion is strongly dependent on the proton motive force, unlike the full-length F₀a protein. These data demonstrate an extensive role of YidC in the assembly of the F₀ sector of the F₁F₀ ATP synthase.     

Dynamic response of UV-absorbing compounds, quantum yield and the xanthophyll cycle to diel changes in UV-B and photosynthetic radiations in an aquatic liverwort

We studied the diel responses of the liverwort Jungermannia exsertifolia subsp. cordifolia to radiation changes under laboratory conditions. The samples were exposed to three radiation regimes: P (only PAR), PA (PAR + UV-A), and PAB (PAR + UV-A + UV-B). The day was divided in four periods: darkness, a first low-PAR period, the high-PAR plus UV period, and a second low-PAR period. After 15 days of culture, we measured photosynthetic pigments, chlorophyll fluorescence and UV-absorbing compounds in the four periods of the day on two consecutive days. With respect to UV-absorbing compounds, we analyzed their global amount (as the bulk UV absorbance of methanolic extracts) and the concentration of seven hydroxycinnamic acid derivatives, both in the soluble (mainly vacuolar) and insoluble (cell wall-bound) fractions of the plant extracts. PAB samples increased the bulk UV absorbance of the soluble and insoluble fractions, and the concentrations of p-coumaroylmalic acid in the soluble fraction and p-coumaric acid in the cell wall. Most of these variables showed significant diel changes and responded within a few hours to radiation changes (more strongly to UV-B), increasing at the end of the period of high-PAR plus UV. F_v/F_m, Φ_PSII, NPQ and the components of the xanthophyll cycle showed significant and quick diel changes in response to high PAR, UV-A and UV-B radiation, indicating dynamic photoinhibition and protection of PSII from excess radiation through the xanthophyll cycle. Thus, the liverwort showed a dynamic protection and acclimation capacity to the irradiance level and spectral characteristics of the radiation received.     

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from TyrZ to P680 and the quinone acceptors QA to QB

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, Q_A and Q_B, and the S₂ state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of Q_A⁻ reoxidation.     

Water deficit in field-grown Gossypium hirsutum primarily limits net photosynthesis by decreasing stomatal conductance,increasing photorespiration,and increasing the ratio of dark respiration to gross photosynthesis

Much effort has been expended to improve irrigation efficiency and drought tolerance of agronomic crops; however, a clear understanding of the physiological mechanisms that interact to decrease source strength and drive yield loss has not been attained. To elucidate the underlying mechanisms contributing to inhibition of net carbon assimilation under drought stress, three cultivars of Gossypium hirsutum were grown in the field under contrasting irrigation regimes during the 2012 and 2013 growing season near Camilla, Georgia, USA. Physiological measurements were conducted on three sample dates during each growing season (providing a broad range of plant water status) and included, predawn and midday leaf water potential (Ψ_PD and Ψ_MD), gross and net photosynthesis, dark respiration, photorespiration, and chlorophyll a fluorescence. End-of-season lint yield was also determined. Ψ_PD ranged from −0.31 to −0.95 MPa, and Ψ_MD ranged from −1.02 to −2.67 MPa, depending upon irrigation regime and sample date. G. hirsutum responded to water deficit by decreasing stomatal conductance, increasing photorespiration, and increasing the ratio of dark respiration to gross photosynthesis, thereby limiting P_N and decreasing lint yield (lint yield declines observed during the 2012 growing season only). Conversely, even extreme water deficit, causing a 54% decline in P_N, did not negatively affect actual quantum yield, maximum quantum yield, or photosynthetic electron transport. It is concluded that P_N is primarily limited in drought-stressed G. hirsutum by decreased stomatal conductance, along with increases in respiratory and photorespiratory carbon losses, not inhibition or down-regulation of electron transport through photosystem II. It is further concluded that Ψ_PD is a reliable indicator of drought stress and the need for irrigation in field-grown cotton.     

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt,p38, and JNK pathways in rabbit articular chondrocytes

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined.     

Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes

The carnitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl₂ and DIDS. The IC₅₀ for HgCl₂ was 0.16 ± 0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9 ± 0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.     

Elucidating the mechanisms of coupled electron transfer and catalytic reactions by protein film voltammetry

Protein film voltammetry, the direct electrochemistry of redox enzymes and proteins, provides precise and comprehensive information on complicated reaction mechanisms. By controlling the driving force for a reaction (using the applied potential) and monitoring the reaction in real time (using the current), it allows thermodynamic and kinetic information to be determined simultaneously. Two challenges are inherent to protein film voltammetry: (i) to adsorb the protein or enzyme in a native and active configuration on the electrode surface, and (ii) to understand and interpret voltammetric results on both a qualitative and quantitative level, allowing mechanistic models to be proposed and rigorous experiments to test these models to be devised. This review focuses on the second of these two challenges. It describes how to use protein film voltammetry to derive mechanistic and biochemically relevant information about redox proteins and enzymes, and how to evaluate and interpret voltammetric results. Selected key studies are described in detail, to illustrate their underlying principles, strategies and physical interpretations.     

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

Antimicrobial peptides (AMPs) are naturally occurring entities with potential as pharmaceutical candidates and/or food additives. They are present in many organisms including bacteria, insects, fish and mammals. While their antimicrobial activity is equipotent with many commercial antibiotics, current limitations are poor pharmacokinetics, stability and potential toxicology issues. Most elicit antimicrobial action via perturbation of bacterial membranes. Consequently, associated cytotoxicity in human cells is reflected by their capacity to lyse erythrocytes. However, more rigorous toxicological assessment of AMPs is required in order to predict potential failure at a later stage of development. We describe a high-content analysis (HCA) screening protocol recently established for determination and prediction of safety in pharmaceutical drug discovery. HCA is a powerful, multi-parameter bioanalytical tool that amalgamates the actions of fluorescence microscopy with automated cell analysis software in order to understand multiple changes in cellular health. We describe the application of HCA in assessing cytotoxicity of the cytolytic α-helical peptide, melittin, and selected structural analogs. The data shows that structural modification of melittin reduces its cytotoxic action and that HCA is suitable for rapidly identifying cytotoxicity.     

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca²⁺-permeable channel currents by ABA or oscillation of the cytosolic free Ca²⁺ concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca²⁺ oscillation in ABA signal pathway of Arabidopsis guard cells.     

Enhancement of YD spin relaxation by the CaMn4 cluster in photosystem II detected at room temperature: A new probe for the S-cycle

The long-lived, light-induced radical Y_D^• of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn₄ cluster, situated ∼ 30 Å away. In this study we report a transient step-change increase in Y_D^• EPR intensity upon the application of a single laser flash to S₁ state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of Y_D^• by the OEC in the S₂ state, as a consequence of the single laser flash turnover. The post-flash decay reflected S₂ → S₁ back-turnover, as confirmed by their correlations with independent measurements of S₂ multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using Y_D^• as a probe.