Electrogenic Properties of the Sodium Pump in a Dynamic Model of Membrane Transport

The general purpose of this theoretical work is to contribute to understand the physiological role of the electrogenic properties of the sodium pump, by studying a dynamic model that integrates diverse processes of ionic and water transport across the plasma membrane. For this purpose, we employ a mathematical model that describes the rate of change of the intracellular concentrations of Na⁺, K⁺ and Cl⁻, of the cell volume, and of the plasma membrane potential (V _m ). We consider the case of a nonexcitable, nonpolarized cell expressing the sodium pump; Na⁺, K⁺, Cl⁻ and water channels, and cotransporters of KCl and NaCl in its plasma membrane. We particularly analyze here the conditions under which the physiological V _m can be generated in a predominantly electrogenic fashion, as a result of the activity of the sodium pump. A major conclusion of this study is that, for the cell model considered, a low potassium permeability is not a sufficient condition for a predominantly electrogenic generation of the V _m by the sodium pump. The presence of an electroneutral exchange of Na⁺ and K⁺ represents a necessary additional requirement. Received: 8 September 1999/Revised: 21 March 2000     

ABA treatment increases both the desiccation tolerance of photosynthesis,and nonphotochemical quenching in the moss Atrichum undulatum

Pulse amplitude modulation fluorescence was used to investigate whether abscisic acid (ABA) pretreatment increases the desiccation tolerance of photosynthesis in the moss Atrichum undulatum. In unstressed plants, ABA pretreatment decreased the F _V/F _m ratio, largely as a result of an increase in F _o. This indicated a reduction in energy transfer between LHCII and PSII, possibly hardening the moss to subsequent stress by reducing the production of the reactive oxygen species near PSII. During desiccation, F ₀, F _m, F _v/F _m, PSII, and NPQ and F ₀ quenching declined in ABA-treated and nontreated mosses. However, during rehydration, F ₀, F _m, F _v/F _m, and PSII recovered faster in ABA-treated plants, suggesting that ABA improved the tolerance of photosystem II to desiccation. NPQ increased upon rehydration in mosses from both treatments, but much more rapidly in ABA-treated plants; during the first hour of rehydration, NPQ was two-fold greater in plants treated with ABA. F ₀quenching followed a similar pattern, indicating that ABA treatment stimulated zeaxanthin-based quenching. The implications of these results for the mechanisms of ABA-induced desiccation tolerance in A. undulatum are discussed.     

Possible involvement of the double-stranded RNA-binding core protein sigmaA in the resistance of avian reovirus to interferon

Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein sigmaA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein sigmaA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins.     

Doxorubicin treatment activates a Z-VAD-sensitive caspase, which causes deltapsim loss, caspase-9 activity, and apoptosis in Jurkat cells

Doxorubicin induces caspase-3 activation and apoptosis in Jurkat cells but inhibition of this enzyme did not prevent cell death, suggesting that another caspase(s) is critically implicated. Western blot analysis of cell extracts indicated that caspases 2, 3, 4, 6, 7, 8, 9, and 10 were activated by doxorubicin. Cotreatment of cells with the caspase inhibitors Ac-DEVD-CHO, Z-VDVAD-fmk, Z-IETD-fmk, and Z-LEHD-fmk alone or in combination, or overexpression of CrmA, prevented many morphological features of apoptosis but not loss of mitochondrial membrane potential (delta(psi)m), phospatidilserine exposure, and cell death. Western blot analysis of cells treated with doxorubicin in the presence of inhibitors allowed elucidation of the sequential order of caspase activation. Z-IETD-fmk or Z-LEHD-fmk, which inhibit caspase-9 activity, blocked the activation of all caspases studied, lamin B degradation, and the development of apoptotic morphology, but not cell death. All morphological and biochemical features of apoptosis, as well as cell death, were prevented by cotreatment of cells with the general caspase inhibitor Z-VAD-fmk or by overexpression of Bcl-2. Doxorubicin cytotoxicity was also blocked by the protein synthesis inhibitor cycloheximide. Delayed addition of Z-VAD-fmk after doxorubicin treatment, but prior to the appearance of cells displaying a low delta(psi)m, prevented cell death. These results, taken together, suggest that the key mediator of doxorubicin-induced apoptosis in Jurkat cells may be an inducible, Z-VAD-sensitive caspase (caspase-X), which would cause delta(psi)m loss, release of apoptogenic factors from mitochondria, and cell death.     

Binding of spin-labeled galactosides to the lactose permease of Escherichia coli

A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.     

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.     

The crystal structure of tetrameric methionine adenosyltransferase from rat liver reveals the methionine-binding site

Most of the transmethylation reactions use the same methyl donor, S-adenosylmethionine (SAM), that is synthesised from methionine and ATP by methionine adenosyltransferase (MAT). In mammals, two MAT enzymes have been detected, one ubiquitous and another liver specific. The liver enzyme exists in two oligomeric forms, a tetramer (MAT I) and a dimer (MAT III), MAT I being the one that shows a higher level of affinity for methionine but a lower SAM synthesis capacity. We have solved the crystal structure of rat liver MAT I at 2.7 A resolution, complexed with a methionine analogue: l-2-amino-4-methoxy-cis-but-3-enoic acid (l-cisAMB). The enzyme consists of four identical subunits arranged in two tight dimers that are related by crystallographic 2-fold symmetry. The crystal structure shows the positions of the relevant cysteine residues in the chain, and that Cys35 and Cys61 are perfectly oriented for forming a disulphide link. This result leads us to propose a hypothesis to explain the control of MAT I/III exchange and hence, the effects observed on activity. We have identified the methionine-binding site into the active-site cavity, for the first time. The l-cisAMB inhibitor is stacked against Phe251 aromatic ring in a rather planar conformation, and its carboxylate group coordinates a Mg(2+), which, in turn, is linked to Asp180. The essential role of the involved residues in MAT activity has been confirmed by site-directed mutagenesis. Phe251 is exposed to solvent and is located in the beginning of the flexible loop Phe251-Ala260 that is connecting the N-terminal domain to the central domain. We postulate that a conformational change may take place during the enzymatic reaction and this is possibly the reason of the unusual two-step mechanism involving tripolyphosphate hydrolysis. Other important mechanistic implications are discussed on the light of the results. Moreover, the critical role that certain residues identified in this study may have in methionine recognition opens further possibilities for rational drug design.     

Exogenous strigolactones impact metabolic profiles and phosphate starvation signalling in roots

Strigolactones (SLs) are important ex-planta signalling molecules in the rhizosphere, promoting the association with beneficial microorganisms, but also affecting plant interactions with harmful organisms. They are also plant hormones in-planta, acting as modulators of plant responses under nutrient-deficient conditions, mainly phosphate (Pi) starvation. In the present work, we investigate the potential role of SLs as regulators of early Pi starvation signalling in plants. A short-term pulse of the synthetic SL analogue 2′-epi-GR24 promoted SL accumulation and the expression of Pi starvation markers in tomato and wheat under Pi deprivation. 2′-epi-GR24 application also increased SL production and the expression of Pi starvation markers under normal Pi conditions, being its effect dependent on the endogenous SL levels. Remarkably, 2′-epi-GR24 also impacted the root metabolic profile under these conditions, promoting the levels of metabolites associated to plant responses to Pi limitation, thus partially mimicking the pattern observed under Pi deprivation. The results suggest an endogenous role for SLs as Pi starvation signals. In agreement with this idea, SL-deficient plants were less sensitive to this stress. Based on the results, we propose that SLs may act as early modulators of plant responses to P starvation.     

New conservation opportunities: Using citizen science in monitoring non-native species in Neotropical region

The combination of highly equipped smartphones, with the increased use of social media, has offered a wide database. Given this, citizen science can be used to record and monitor non-native fish fauna, target new samples and collaborate with monitoring occurrences in new areas. We aimed to demonstrate the efficiency of social media in citizen science as a tool to cooperate with monitoring studies of non-native species. Consequently, we determined the occurrence points of S. brasiliensis in the Iguaçu River basin, indicating sites of greatest occurrence and analyzing the impact of the invasion on the native fauna of the basin. Files and information available on the YouTube^® and Facebook^® media platforms were used as data, was carried out from April 2019 to April 2020. The results were 40 records, 22 videos obtained from Youtube, and seven videos and 11 photos from Facebook, the oldest record was from April 2013, while the largest number of posts was in 2016. Fish records available from online platforms can reveal the occurrence and progressive dispersion of species, in the context of biological invasions, these tools can be of great value in studies that aim to follow the progress of introduced species, contributing by helping to direct new sampling programs and corroborating the occurrence of species in new areas in conjunction with standard monitoring programs. Based on citizen science records, it was possible to update the range of occurrence of the non-native S. brasiliensis in the Iguaçu River basin, cooperating with scientific knowledge. Innovative monitoring and control measures are necessary to deal with invasive species, with citizen science proving to be competent for determining the occurrence of species and showing promise in the entire field of ichthyology.