RESUMEN
We report the first dark matter search results from XENON1T, a â¼2000-kg-target-mass dual-phase (liquid-gas) xenon time projection chamber in operation at the Laboratori Nazionali del Gran Sasso in Italy and the first ton-scale detector of this kind. The blinded search used 34.2 live days of data acquired between November 2016 and January 2017. Inside the (1042±12)-kg fiducial mass and in the [5,40] keV_{nr} energy range of interest for weakly interacting massive particle (WIMP) dark matter searches, the electronic recoil background was (1.93±0.25)×10^{-4} events/(kg×day×keV_{ee}), the lowest ever achieved in such a dark matter detector. A profile likelihood analysis shows that the data are consistent with the background-only hypothesis. We derive the most stringent exclusion limits on the spin-independent WIMP-nucleon interaction cross section for WIMP masses above 10 GeV/c^{2}, with a minimum of 7.7×10^{-47} cm^{2} for 35-GeV/c^{2} WIMPs at 90% C.L.
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We report on a search for electronic recoil event rate modulation signatures in the XENON100 data accumulated over a period of 4 yr, from January 2010 to January 2014. A profile likelihood method, which incorporates the stability of the XENON100 detector and the known electronic recoil background model, is used to quantify the significance of periodicity in the time distribution of events. There is a weak modulation signature at a period of 431_{-14}^{+16} day in the low energy region of (2.0-5.8) keV in the single scatter event sample, with a global significance of 1.9σ; however, no other more significant modulation is observed. The significance of an annual modulation signature drops from 2.8σ, from a previous analysis of a subset of this data, to 1.8σ with all data combined. Single scatter events in the low energy region are thus used to exclude the DAMA/LIBRA annual modulation as being due to dark matter electron interactions via axial vector coupling at 5.7σ.
RESUMEN
We have searched for periodic variations of the electronic recoil event rate in the (2-6) keV energy range recorded between February 2011 and March 2012 with the XENON100 detector, adding up to 224.6 live days in total. Following a detailed study to establish the stability of the detector and its background contributions during this run, we performed an unbinned profile likelihood analysis to identify any periodicity up to 500 days. We find a global significance of less than 1σ for all periods, suggesting no statistically significant modulation in the data. While the local significance for an annual modulation is 2.8σ, the analysis of a multiple-scatter control sample and the phase of the modulation disfavor a dark matter interpretation. The DAMA/LIBRA annual modulation interpreted as a dark matter signature with axial-vector coupling of weakly interacting massive particles to electrons is excluded at 4.8σ.
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Bisphenol A (BPA), a widespread man-made chemical classified as an endocrine disruptor, is increasingly considered as a major cause of concern for human health. Chlorine present in drinking water may react with BPA to form chlorinated derivatives (ClxBPA), which have demonstrated a heightened level of estrogenic activity. If many epidemiological studies report that more than 90% of people have detectable BPA levels in their urine, then no such study has been undertaken regarding ClxBPA. The purpose of this work is to propose a highly sensitive and accurate analytical method adapted to large-scale biomonitoring studies aimed at assessing exposure to BPA and ClxBPA through the use of human urine. To achieve this, we have comprehensively validated a method using salting-out assisted liquid/liquid extraction (SALLE) coupled to UPLC-MS/MS and isotope dilution quantification, to measure unconjugated BPA and ClxBPA in human urine according to the accepted guidelines. Deutered BPA as well as deutered 2,2'-DCBPA was used as internal standards. The matrix calibration curve ranged from 0.05 to 1.60 ng mL(-1) and from 0.5 to 16.0 ng mL(-1) for ClxBPA and BPA respectively, and provided good linearity (r²>0.99). This method was precise (the intra- and inter-day coefficients of variation were <20% at three different concentrations: 0.05 ng mL(-1), 0.2 ng mL(-1), 0.8 ng mL(-1) and 0.5 ng mL(-1), 2 ng mL(-1), 8 ng mL(-1) for ClxBPA and BPA, respectively) and accurate (bias ranged from -13% to +12%). The limit of quantification, validated at 0.05 ng mL(-1) and 0.5 ng mL(-1) for ClxBPA and BPA respectively when using 300 µL of urine, was found to be suitable for the concentration existing in real samples. The matrix effect and the BPA cross-contamination were also investigated in this study. The analytical method developed in this study is in accordance with the requirements applicable to biomonitoring of BPA and ClxBPA in human urine.
Asunto(s)
Compuestos de Bencidrilo/orina , Cloro/análisis , Cromatografía Líquida de Alta Presión , Fenoles/orina , Espectrometría de Masas en Tándem , Urinálisis/normas , Orina/química , Calibración , Técnicas de Química Analítica , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We present new experimental constraints on the elastic, spin-dependent WIMP-nucleon cross section using recent data from the XENON100 experiment, operated in the Laboratori Nazionali del Gran Sasso in Italy. An analysis of 224.6 live days×34 kg of exposure acquired during 2011 and 2012 revealed no excess signal due to axial-vector WIMP interactions with 129Xe and 131Xe nuclei. This leads to the most stringent upper limits on WIMP-neutron cross sections for WIMP masses above 6 GeV/c², with a minimum cross section of 3.5×10(-40) cm² at a WIMP mass of 45 GeV/c², at 90% confidence level.
RESUMEN
We report on a search for particle dark matter with the XENON100 experiment, operated at the Laboratori Nazionali del Gran Sasso for 13 months during 2011 and 2012. XENON100 features an ultralow electromagnetic background of (5.3 ± 0.6) × 10(-3) events/(keV(ee) × kg × day) in the energy region of interest. A blind analysis of 224.6 live days × 34 kg exposure has yielded no evidence for dark matter interactions. The two candidate events observed in the predefined nuclear recoil energy range of 6.6-30.5 keV(nr) are consistent with the background expectation of (1.0 ± 0.2) events. A profile likelihood analysis using a 6.6-43.3 keV(nr) energy range sets the most stringent limit on the spin-independent elastic weakly interacting massive particle-nucleon scattering cross section for weakly interacting massive particle masses above 8 GeV/c(2), with a minimum of 2 × 10(-45) cm(2) at 55 GeV/c(2) and 90% confidence level.
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Adhesion molecules can improve hematopoietic cell survival; however, their role in leukemic cell resistance to drug-induced apoptosis is poorly documented. The CD44 adhesion molecule is strongly expressed on acute myeloid leukemia (AML) blasts. Using 2 myeloid cell lines, HL60 and NB4, evidence is presented that prior incubation with the CD44-specific monoclonal antibody (mAb) A3D8, reported to induce differentiation of AML blasts, significantly decreases apoptosis induced by 3 drugs used in AML chemotherapy: daunorubicin (DNR), mitoxantrone, and etoposide. In addition, in HL60 cells, CD44 ligation with A3D8 mAb fully abrogates the DNR-triggered generation of ceramide, a lipid second messenger involved in the DNR apoptotic signaling pathway. Moreover, results show that the A3D8 mAb and Bcl-2 additively inhibit DNR-induced apoptosis in HL60 cells overexpressing Bcl-2. These results suggest that, to eradicate AML blasts, the differentiation-inducing anti-CD44 mAb A3D8 should not be administered prior to apoptosis-inducing drugs.
Asunto(s)
Apoptosis , Receptores de Hialuranos , Leucemia Mieloide/patología , Moléculas de Adhesión Celular , Humanos , Leucemia Mieloide/inmunología , Células Tumorales CultivadasRESUMEN
Since the beginning of the 1990s, our knowledge of the protein equipment of plant membranes progresses at an accelerating pace, owing to the irruption of molecular biology tools and genetics strategies in plant biology. Map-based cloning strategies and exploration of EST databases rapidly enrich the catalog of cDNA or gene sequences expected to code for membrane proteins. The accumulation of 'putative' membrane proteins reinforces the need for structural, functional and physiological information. Indeed, ambiguities often exist concerning the association to a membrane, the membrane identity and the topology of the protein inserted in the membrane. The combination of directed mutagenesis and heterologous expression of plant genes in various systems and plant reverse genetics has opened the possibility to study molecular and physiological functions. This review will emphasize how these tools have been essential for the exciting recent discoveries on plant terminal membrane proteins. These discoveries concern a variety of transport systems for ions, organic solutes including auxin, water channels, a large collection of systems suspected to act as receptors of chemical signals, proteins thought to control vesicle trafficking and enzymatic systems.
Asunto(s)
Proteínas de la Membrana/fisiología , Proteínas de Plantas/fisiología , Animales , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de Plantas/metabolismoAsunto(s)
Agricultura , Ingestión de Alimentos , Abastecimiento de Alimentos , Salud Pública , Factores Socioeconómicos , Agricultura/economía , Agricultura/educación , Agricultura/historia , Agricultura/legislación & jurisprudencia , Dieta/economía , Dieta/etnología , Dieta/historia , Dieta/psicología , Ingestión de Alimentos/etnología , Ingestión de Alimentos/fisiología , Ingestión de Alimentos/psicología , Alimentos/economía , Alimentos/historia , Abastecimiento de Alimentos/economía , Abastecimiento de Alimentos/historia , Abastecimiento de Alimentos/legislación & jurisprudencia , Francia/etnología , Agencias Gubernamentales/economía , Agencias Gubernamentales/historia , Agencias Gubernamentales/legislación & jurisprudencia , Historia del Siglo XX , Estilo de Vida/etnología , Salud Pública/economía , Salud Pública/educación , Salud Pública/historia , Salud Pública/legislación & jurisprudencia , Política Pública/economía , Política Pública/historia , Política Pública/legislación & jurisprudencia , Estadística como Asunto/economía , Estadística como Asunto/educación , Estadística como Asunto/historia , Estadística como Asunto/legislación & jurisprudenciaRESUMEN
To determine if the ATP sulfurylase reaction is a regulatory step for the SO4(2-)-assimilation pathway in plants, an Arabidopsis thaliana ATP sulfurylase cDNA, APS2, was fused to the 35S promoter of the cauliflower mosaic virus and introduced by Agrobacterium tumefaciens-mediated transformation into isolated Bright Yellow 2 tobacco (Nicotiana tabacum) cells. The ATP sulfurylase activity in transgenic cells was 8-fold that in control cells, and was correlated with the expression of a specific polypeptide revealed by western analysis using an anti-ATP sulfurylase antibody. The molecular mass of this polypeptide agreed with that for the overexpressed mature protein. ATP sulfurylase overexpression had no effect on [35S]SO4(2-) influx or ATP sulfurylase activity regulation by S availability, except that ATP sulfurylase activity variations in response to S starvation in transgenic cells were 8 times higher than in the wild type. There were also no differences in cell growth or sensitivity to SeO4(2-) (a toxic SO4(2-) analog) between transgenic and wild-type cells. We propose that in Bright Yellow 2 tobacco cells, the ATP sulfurylase derepression by S deficiency may involve a posttranscriptional mechanism, and that the ATP sulfurylase abundance is not limiting for cell metabolism.
Asunto(s)
Arabidopsis/enzimología , Sulfato Adenililtransferasa/metabolismo , Sulfatos/metabolismo , Arabidopsis/genética , División Celular , Células Cultivadas , Clonación Molecular , Expresión Génica , Plantas Modificadas Genéticamente , Plantas Tóxicas , Ácido Selénico , Compuestos de Selenio/metabolismo , Sulfato Adenililtransferasa/genética , NicotianaRESUMEN
Proteins from phase-partitioned corn root plasma membrane were reconstituted into soybean lipids/egg PC (8:2, w:w) using deoxycholate and rapid gel filtration to eliminate the detergent. All (H+)ATPase molecules were inside-out reinserted and the initial activity was totally recovered in an homogeneous vesicle preparation. In addition, membrane tightness greatly increased, as shown by the size and stability of the response of the fluorescent membrane potential probe (oxonol VI) to an imposed K+ diffusion gradient. Consequently, the H(+)-pumping activity of the (H+)ATPase, monitored with the fluorescent pH probe (ACMA), increased 20-fold after reconstitution. A protein-mediated passive transport of nitrate was first demonstrated by the ability of NO3- to electrically short-circuit the (H+)ATPase in plasma membrane vesicles and not in liposomes containing only the purified enzyme. The passive transport was saturable (K(m) approximately 5 mM), thermolabile, inhibited by the arginine reagent phenylglyoxal, and selective (NO3- > I- approximately ClO3- approximately Br- > Cl- approximately NO2- > Iminodiacetate approximately SO4(2-)). Passive NO3- transport was also determined, independently of the (H+)ATPase, from the NO3(-)-dependent augmentation of the dissipation rate of imposed diffusion potentials. This second transport assay gave similar K(m) for NO3- and should be suitable to continue the functional and biochemical characterization of the NO3- transport system.
Asunto(s)
Vesículas Cubiertas/metabolismo , Nitratos/metabolismo , Raíces de Plantas/metabolismo , Transporte Biológico , Proteínas de la Membrana/metabolismo , Raíces de Plantas/citología , ATPasas de Translocación de Protón/metabolismo , Valinomicina , Zea maysRESUMEN
We have previously demonstrated that daunorubicin (DNR) induces apoptosis in some leukemic myeloid cell lines. We investigated a potential protective role for Bcl-2 in apoptosis induced by DNR in two leukemic cell lines, one myeloid and one lymphoid, overexpressing the anti-apoptotic gene Bcl-2. Parental cells treated with DNR exhibited classical features of apoptosis 6 h after drug exposure, all the cells being dead after 30-48 h. In contrast, overexpression of Bcl-2 significantly delayed, but did not prevent the occurrence of DNR-induced apoptosis, with no surviving cells 96 h after drug exposure. To elucidate the mechanism of the protection mediated by Bcl-2, we explored the signaling pathway which initiates DNR-induced apoptosis. In this report, we show that, in both the myeloid and lymphoid parental cell lines, DNR triggered a sphingomyelin (SM) hydrolysis after 10-15 min with a concomitant ceramide generation. Moreover, exogenous ceramide induced DNA fragmentation in these cells, with levels similar to those observed with DNR treatment. In contrast, Bcl-2 overexpression protected the cells against apoptosis induced by ceramide treatment, without preventing the early SM hydrolysis nor the ceramide generation in these cells. Our results strongly suggest that Bcl-2-mediated protection of DNR-induced apoptosis is effected downstream of the SM-ceramide signaling pathway.
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Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Ceramidas/metabolismo , Daunorrubicina/farmacología , Células HL-60/metabolismo , Células HL-60/patología , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patología , ADN de Neoplasias/metabolismo , Células HL-60/efectos de los fármacos , Humanos , Hidrólisis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Transducción de Señal/fisiología , Transducción Genética , Proteína X Asociada a bcl-2RESUMEN
An Arabidopsis thaliana ATP sulfurylase cDNA (ASA1), encoding a putative chloroplastic isoform, has been cloned by functional complementation of a Saccharomyces cerevisiae (met3) ATP sulfurylase mutant which also has a poor sulfate transport capacity. Homologous complementation of the yeast mutant with the ATP sulfurylase gene restores both ATP sulfurylase function and sulfate transport. Heterologous complementation restores only ATP sulfurylase function as demonstrated by low [35S]sulfate influx measurements and selenate resistance. A structural relationship between ATP sulfurylase and sulfate membrane transporters in yeast is proposed. The sequence of ASA1 is homologous to deduced plant and animal ATP sulfurylase sequences. Analyses indicate a potential tyrosine phosphorylation site which is unique to higher eukaryote sequences. ASA1 is specified by a single copy gene that is part of a multigene family in A. thaliana. At least two ASA1 copies are found in Brassica napus plants. ASA1 transcripts were found in all organs examined, with the highest transcript abundance and ATP sulfurylase activity in leaves or cotyledons. Absence of sulfate from culture media transiently increased B. napus transcript abundance, indicating that initially, the response to sulfate deprivation is transcriptionally regulated.
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Arabidopsis/enzimología , Familia de Multigenes , Azufre/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Brassica/metabolismo , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Hojas de la Planta/enzimología , Raíces de Plantas/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
AKT1, a putative inwardly directed K+ channel of Arabidopsis, restores long-term potassium uptake in a yeast mutant defective in K+ absorption. In this paper, the expression pattern of the gene encoding AKT1 is described. Northern blots indicate that AKT1 transcripts are preferentially accumulated in Arabidopsis roots. Owing to the difficulties in producing large quantities of Arabidopsis roots under hydroponic conditions, experiments were undertaken with Brassica napus, a related species. Potassium starvation experiments on B. napus plants show that changes in the K+ status of the organs do not modify AKT1 mRNA accumulation. Western blot analysis of B. napus proteins confirms the presence of AKT1 at the root plasma membrane. Tissue-specific expression directed by the Arabidopsis AKT1 gene promoter was investigated by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing an AKT1-GUS gene fusion. As determined by fluorimetric and histochemical tests, the AKT1 promoter directs preferential expression in the peripheral cell layers of root mature regions. The discrete activity found in leaves relates to leaf primordia and to small groups of cells, hydathodes, found on toothed margins of the Arabidopsis leaf lamina. These data are discussed with regard to a possible role of AKT1 in K+ nutrition of plants.
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Proteínas de Arabidopsis , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas de Plantas/metabolismo , Plantas/genética , Canales de Potasio/metabolismo , Potasio/metabolismo , Arabidopsis/química , Arabidopsis/genética , Secuencia de Bases , Brassica/química , Brassica/genética , Membrana Celular/química , Datos de Secuencia Molecular , Raíces de Plantas/química , Plantas/química , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , ARN de Planta/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Distribución TisularRESUMEN
It has been claimed that the inorganic pyrophosphatase (PPase) of the plant vacuolar membrane transports K+ in addition to H+ in intact vacuoles (Davies, J. M., Poole, R. J., Rea, P. A., and Sanders, D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11701-11705). Since this was not confirmed using the purified and reconstituted PPase consisting of a 75-kDa polypeptide (Sato, M.H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., and Yoshida, M. (1994) J. Biol. Chem. 269, 6725-6728), these authors proposed that K+ transport by the PPase is dependent on its association with other membrane components lost during purification. We have examined the hypothesis of K+ translocation by the PPase using native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for K+, H+, and membrane potential. This material contained a high proportion of right-side-out, tightly sealed vesicles, exhibiting high PPase activity which was strongly stimulated by uncouplers and K+. Proton pumping occurred in response to pyrophosphate addition in the absence of K+. No K+ incorporation into the vesicles could be observed after PPase energization in the presence of K+, although H+ transport was highly stimulated. The hydrolytic activity was stimulated by a protonophore and by a H+/K+ exchanger but not by the K+ ionophore valinomycin. No evidence could be obtained supporting the operation of an endogenous K+/H+ exchanger capable to dissipate the putative active K+ flux generated by the PPase. We conclude that PPase in native vacuolar membrane vesicles does not transport K+.
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Hidrógeno/metabolismo , Potenciales de la Membrana , Plantas/enzimología , Potasio/metabolismo , Pirofosfatasas/metabolismo , Vacuolas/metabolismo , Activación Enzimática , Colorantes Fluorescentes , Pirofosfatasa Inorgánica , Membranas Intracelulares/metabolismo , Transporte Iónico , ProtonesRESUMEN
The hypothesis that the binding of an antibody to a membrane protein is likely to prevent the reconstitution of the protein into liposomes was checked, by using the plant plasma membrane H(+)-ATPase (EC 3.6.1.35) as a model system, and two reconstitution procedures: spontaneous insertion (SI) of purified H(+)-ATPase into preformed liposomes, and a detergent-mediated reconstitution (DMR) procedure allowing the reconstitution of the whole membrane protein content. Nine monoclonal antibodies (MABs) raised against H(+)-ATPase were tested. None affected the functioning of the enzyme reconstituted in liposomes, suggesting that the probability to obtain an inhibitory MAB is low. Five MABs inhibited its SI, and seven inhibited its reconstitution in the DMR procedure. These results indicate that it is possible to screen antibodies directed against membrane protein, by making use of their ability to inhibit the reconstitution of these proteins.
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Anticuerpos Monoclonales/química , Proteínas de la Membrana/química , Proteínas de Plantas/química , Proteínas de la Membrana/inmunología , Proteínas de Plantas/inmunologíaRESUMEN
Liposomes of egg PC/PG (8:2, mol/mol) were multilabelled with PBFI, pyranine and oxonol VI, fluorescent probes for, respectively, K+, H+ and membrane potential. Monitoring fluorescence with a multichannel photoncounting spectrofluorometer during K+ filling experiments allowed to measure K+ influx, the associated H+ efflux and the membrane potential, continuously and simultaneously. The proton net efflux quantitatively mirrored the K+ net influx. The rate of the K+/H+ exchange diminished progressively as a quasi-equilibrium was reached for both K+ and H+. In the presence of valinomycin, the measured membrane potential during the K+ filling actually corresponded to the Nernst potential calculated from the observed K+ gradient. In the absence of valinomycin, it corresponded to the Nernst potential calculated from the observed H+ gradient. In the latter case, the permeability coefficient of liposomes to K+, calculated from the Goldman-Hodgkin-Katz relation, was 6.10(-13) m s-1. The selectivity sequence for alkali cations of liposomes was determined from the measured H+ efflux associated to the influx of the different cations. The selectivity sequence corresponded to the series VI of Eisenman, suggesting interaction of the cation with an anionic field of intermediate strength.
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Cationes/análisis , Colorantes Fluorescentes , Liposomas , Arilsulfonatos , Benzofuranos , Éteres Cíclicos , Concentración de Iones de Hidrógeno , Matemática , Potenciales de la Membrana , Permeabilidad , Cloruro de Potasio , Protones , ValinomicinaRESUMEN
In soybean (Glycine max L. Merr. cv Kingsoy), NO(3) (-) assimilation in leaves resulted in production and transport of malate to roots (B Touraine, N Grignon, C Grignon [1988] Plant Physiol 88: 605-612). This paper examines the significance of this phenomenon for the control of NO(3) (-) uptake by roots. The net NO(3) (-) uptake rate by roots of soybean plants was stimulated by the addition of K-malate to the external solution. It was decreased when phloem translocation was interrupted by hypocotyl girdling, and partially restored by malate addition to the medium, whereas glucose was ineffective. Introduction of K-malate into the transpiration stream using a split root system resulted in an enrichment of the phloem sap translocated back to the roots. This treatment resulted in an increase in both NO(3) (-) uptake and C excretion rates by roots. These results suggest that NO(3) (-) uptake by roots is dependent on the availability of shoot-borne, phloem-translocated malate. Shoot-to-root transport of malate stimulated NO(3) (-) uptake, and excretion of HCO(3) (-) ions was probably released by malate decarboxylation. NO(3) (-) uptake rate increased when the supply of NO(3) (-) to the shoot was increased, and decreased when the activity of nitrate reductase in the shoot was inhibited by WO(4) (2-). We conclude that in situ, NO(3) (-) reduction rate in the shoot may control NO(3) (-) uptake rate in the roots via the translocation rate of malate in the phloem.
RESUMEN
A membrane polypeptide involved in K+ transport in a higher plant was cloned by complementation of a yeast mutant defective in K+ uptake with a complementary DNA library from Arabidopsis thaliana. A 2.65-kilobase complementary DNA conferred ability to grow on media with K+ concentration in the micromolar range and to absorb K+ (or 86Rb+) at rates similar to those in wild-type yeast. The predicted amino acid sequence (838 amino acids) has three domains: a channel-forming region homologous to animal K+ channels, a cyclic nucleotide-binding site, and an ankyrin-like region.
Asunto(s)
Proteínas de Arabidopsis , Clonación Molecular , Proteínas de Plantas/genética , Plantas/genética , Canales de Potasio/genética , Potasio/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Transporte Biológico , Southern Blotting , Proteínas Portadoras/química , Proteínas Portadoras/genética , ADN/genética , Desoxirribonucleasa EcoRI , Expresión Génica , Cinética , Datos de Secuencia Molecular , Proteínas de Plantas/química , Canales de Potasio/química , Homología de Secuencia de Ácido NucleicoRESUMEN
The uptake of sulphate into roots of barley seedlings is highly sensitive to phenylglyoxal (PhG), an arginine-binding reagent. Uptake was inhibited by >80% by a 1-h pre-treatment of roots with 0.45 mol · m(-3) PhG. Inhibition was maximal in pre-treatment solutions buffered between pH 4.5 and 6.5. Phosphate uptake, measured simultaneously by double-labelling uptake solutions with (32)P and (35)S, was less susceptible to inhibition by PhG, particularly at pH <6.5, and was completely insensitive to the less permeant reagent p-hydroxyphenylglyoxal (OH-PhG) administered at 1 mol · m(-3) at pH at 5.0 or 8.2; sulphate uptake was inhibited in -S plants by 90% by OH-PhG-treatment. Root respiration in young root segments was unaffected by OH-PhG pre-treatment for 1 h and inhibited by only 17% after 90 min pre-treatment. The uptake of both ions was inhibited by the dithiol-specific reagent, phenylarsine oxide even after short exposures (0.5-5.0 min). Sulphate uptake was more severely inhibited than that of phosphate, but in both cases inhibition could be substantially reversed by 5 min washing of treated roots by 5 mol · m(-3) dithioerythritol. After longer pre-treatment (50 min) with phenylarsine oxide, inhibition of the ion fluxes was not relieved by washing with dithioerythritol. Inhibition of sulphate influx by PhG was completely reversed by washing the roots for 24 h with culture solution lacking the inhibitor. The reversal was dependent on protein synthesis; less than 20% recovery was seen in the presence of 50 mmol · m(-3) cycloheximide. Sulphate uptake declined rapidly when -S roots were treated with cycloheximide. In the same roots the phosphate influx was little affected, small significant inhibitions being seen only after 4 h of treatment. Respiration was depressed by only 20% in apical and by 31% in basal root segments by cycloheximide pre-treatment for 2 h. Similar rates of collapse of the sulphate uptake and insensitivity of phosphate uptake were seen when protein synthesis was inhibited by azetidine carboxylic acid, p-fluorophenylalanine and puromycin. Considering the effects of all of the protein-synthesis inhibitors together leads to the conclusion that the sulphate transporter itself, or some essential sub-component of the uptake system, turns over rapidly with a half-time of about 2.5 h. The turnover of the phosphate transporter is evidently much slower. The results are discussed in relation to strategies for identifying the transport proteins and to the regulation of transporter activity during nutrient stress.