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1.
Geophys Res Lett ; 46(24): 14826-14835, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33012881

RESUMO

During winter in the mid-latitudes, photochemical oxidation is significantly slower than in summer and the main radical oxidants driving formation of secondary pollutants, such as fine particulate matter and ozone, remain uncertain, owing to a lack of observations in this season. Using airborne observations, we quantify the contribution of various oxidants on a regional basis during winter, enabling improved chemical descriptions of wintertime air pollution transformations. We show that 25-60% of NOx is converted to N2O5 via multiphase reactions between gas-phase nitrogen oxide reservoirs and aerosol particles, with ~93% reacting in the marine boundary layer to form >2.5 ppbv ClNO2. This results in >70% of the oxidizing capacity of polluted air during winter being controlled, not by typical photochemical reactions, but from these multiphase reactions and emissions of volatile organic compounds, such as HCHO, highlighting the control local anthropogenic emissions have on the oxidizing capacity of the polluted wintertime atmosphere.

2.
Atmos Chem Phys ; 16(9): 5969-5991, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-29681921

RESUMO

Formation of organic nitrates (RONO2) during oxidation of biogenic volatile organic compounds (BVOCs: isoprene, monoterpenes) is a significant loss pathway for atmospheric nitrogen oxide radicals (NOx), but the chemistry of RONO2 formation and degradation remains uncertain. Here we implement a new BVOC oxidation mechanism (including updated isoprene chemistry, new monoterpene chemistry, and particle uptake of RONO2) in the GEOS-Chem global chemical transport model with ∼25 × 25 km2 resolution over North America. We evaluate the model using aircraft (SEAC4RS) and ground-based (SOAS) observations of NOx, BVOCs, and RONO2 from the Southeast US in summer 2013. The updated simulation successfully reproduces the concentrations of individual gas- and particle-phase RONO2 species measured during the campaigns. Gas-phase isoprene nitrates account for 25-50% of observed RONO2 in surface air, and we find that another 10% is contributed by gas-phase monoterpene nitrates. Observations in the free troposphere show an important contribution from long-lived nitrates derived from anthropogenic VOCs. During both campaigns, at least 10% of observed boundary layer RONO2 were in the particle phase. We find that aerosol uptake followed by hydrolysis to HNO3 accounts for 60% of simulated gas-phase RONO2 loss in the boundary layer. Other losses are 20% by photolysis to recycle NOx and 15% by dry deposition. RONO2 production accounts for 20% of the net regional NOx sink in the Southeast US in summer, limited by the spatial segregation between BVOC and NOx emissions. This segregation implies that RONO2 production will remain a minor sink for NOx in the Southeast US in the future even as NOx emissions continue to decline.

3.
Atmos Chem Phys ; 16(3): 1603-1618, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32742280

RESUMO

Isoprene emitted by vegetation is an important precursor of secondary organic aerosol (SOA), but the mechanism and yields are uncertain. Aerosol is prevailingly aqueous under the humid conditions typical of isoprene-emitting regions. Here we develop an aqueous-phase mechanism for isoprene SOA formation coupled to a detailed gas-phase isoprene oxidation scheme. The mechanism is based on aerosol reactive uptake coefficients (γ) for water-soluble isoprene oxidation products, including sensitivity to aerosol acidity and nucleophile concentrations. We apply this mechanism to simulation of aircraft (SEAC4RS) and ground-based (SOAS) observations over the Southeast US in summer 2013 using the GEOS-Chem chemical transport model. Emissions of nitrogen oxides (NOx ≡ NO + NO2) over the Southeast US are such that the peroxy radicals produced from isoprene oxidation (ISOPO2) react significantly with both NO (high-NOx pathway) and HO2 (low-NOx pathway), leading to different suites of isoprene SOA precursors. We find a mean SOA mass yield of 3.3 % from isoprene oxidation, consistent with the observed relationship of total fine organic aerosol (OA) and formaldehyde (a product of isoprene oxidation). Isoprene SOA production is mainly contributed by two immediate gas-phase precursors, isoprene epoxydiols (IEPOX, 58% of isoprene SOA) from the low-NOx pathway and glyoxal (28%) from both low- and high-NOx pathways. This speciation is consistent with observations of IEPOX SOA from SOAS and SEAC4RS. Observations show a strong relationship between IEPOX SOA and sulfate aerosol that we explain as due to the effect of sulfate on aerosol acidity and volume. Isoprene SOA concentrations increase as NOx emissions decrease (favoring the low-NOx pathway for isoprene oxidation), but decrease more strongly as SO2 emissions decrease (due to the effect of sulfate on aerosol acidity and volume). The US EPA projects 2013-2025 decreases in anthropogenic emissions of 34% for NOx (leading to 7% increase in isoprene SOA) and 48% for SO2 (35% decrease in isoprene SOA). Reducing SO2 emissions decreases sulfate and isoprene SOA by a similar magnitude, representing a factor of 2 co-benefit for PM2.5 from SO2 emission controls.

4.
Environ Sci Technol ; 47(21): 12566-74, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24088179

RESUMO

Airborne measurements of aerosol composition and gas phase compounds over the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico in June 2010 indicated the presence of high concentrations of secondary organic aerosol (SOA) formed from organic compounds of intermediate volatility. In this work, we investigated SOA formation from South Louisiana crude oil vapors reacting with OH in a Potential Aerosol Mass flow reactor. We use the dependence of evaporation time on the saturation concentration (C*) of the SOA precursors to separate the contribution of species of different C* to total SOA formation. This study shows consistent results with those at the DWH oil spill: (1) organic compounds of intermediate volatility with C* = 10(5)-10(6) µg m(-3) contribute the large majority of SOA mass formed, and have much larger SOA yields (0.37 for C* = 10(5) and 0.21 for C* = 10(6) µg m(-3)) than more volatile compounds with C*≥10(7) µg m(-3), (2) the mass spectral signature of SOA formed from oxidation of the less volatile compounds in the reactor shows good agreement with that of SOA formed at DWH oil spill. These results also support the use of flow reactors simulating atmospheric SOA formation and aging.


Assuntos
Aerossóis/química , Poluentes Atmosféricos/química , Petróleo/análise , Gases , Golfo do México , Laboratórios , Compostos Orgânicos/análise , Poluição por Petróleo , Volatilização
5.
Science ; 337(6099): 1210-2, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22955831

RESUMO

Laboratory studies have established a number of chemical pathways by which nitrogen oxides (NO(x)) affect atmospheric organic aerosol (OA) production. However, these effects have not been directly observed in ambient OA. We report measurements of particulate organic nitrates in Bakersfield, California, the nighttime formation of which increases with NO(x) and is suppressed by high concentrations of organic molecules that rapidly react with nitrate radical (NO(3))--evidence that multigenerational chemistry is responsible for organic nitrate aerosol production. This class of molecules represents about a third of the nighttime increase in OA, suggesting that most nighttime secondary OA is due to the NO(3) product of anthropogenic NO(x) emissions. Consequently, reductions in NO(x) emissions should reduce the concentration of organic aerosol in Bakersfield and the surrounding region.

6.
J Microsc ; 231(2): 349-57, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18778432

RESUMO

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.


Assuntos
Células/química , Microscopia Confocal/métodos , Cebolas/química , Proteínas de Plantas/análise , Plasmodesmos/química , Proteínas de Fluorescência Verde/análise , Proteínas Recombinantes de Fusão/análise
7.
FEBS Lett ; 537(1-3): 96-100, 2003 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-12606038

RESUMO

Divalent metal binding proteins in the Arabidopsis mitochondrial proteome were analysed by mobility shifts in the presence of divalent cations during two-dimensional diagonal sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Tandem mass spectrometry and searches of the predicted Arabidopsis protein dataset were used in an attempt to identify 34 of the proteins which shifted. This analysis identified a total of 23 distinct protein spots as the products of at least 11 different Arabidopsis genes. A series of proteins known to be divalent cation-binding proteins, or to catalyse divalent cation-dependent reactions, were identified. These included: succinyl CoA ligase beta subunit, Mn-superoxide dismutase (SOD), an Fe-S centred component of complex I and the REISKE iron-sulphur protein of the b/c(1) complex. A further set of four proteins of known function but without known divalent binding properties were also identified: the Vb subunit of cytochrome c oxidase, a subunit of ATP synthase (orfB), the acyl carrier protein, and the translocase of the outer membrane (TOM20). Three other proteins, of unknown function, were also found to shift in the presence of divalent cations. This approach has broad application for the identification of sub-proteomes based on the metal interaction of polypeptides.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Cátions Bivalentes/metabolismo , Metais/metabolismo , Mitocôndrias/metabolismo , Proteoma/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Biologia Computacional , Genes de Plantas , Cinética , Espectrometria de Massas
8.
Plant J ; 32(6): 891-904, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12492832

RESUMO

Treatment of Arabidopsis cell culture for 16 h with H2O2, menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatments indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased >3-fold in H2O2/menadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H2O2 as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O2 consumption by mitochondria was significantly decreased by H2O2 treatment.


Assuntos
Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Antimicina A/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Células Cultivadas , Eletroforese em Gel Bidimensional , Endopeptidases/biossíntese , Indução Enzimática/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Oxigênio/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Proteínas de Plantas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vitamina K 3/farmacologia
9.
J Gene Med ; 3(5): 458-67, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11601759

RESUMO

BACKGROUND: DISC-hGMCSF is a gH-deleted HSV-2 based vector expressing human GM-CSF that is being developed for cancer immunotherapy. To support first clinical use, a range of preclinical safety studies were performed using DISC-hGMCSF in addition to DISC-murine-GMCSF and the backbone vector, TA-HSV. METHODS: The toxicity of the DISC vectors was assessed by repeated dose, neurovirulence and neuroinvasiveness studies in mice, and by safety studies in rabbits, guinea pigs and athymic nude mice. Studies were also conducted to determine whether the vector could establish latency in local ganglia in mice following intradermal injection, and whether it could reactivate from the latent state. The vector biodistribution following intravenous administration was also investigated in mice, using PCR to detect vector DNA. RESULTS: The DISC vectors were essentially non-toxic in all the systems studied. No adverse reactions were seen in mice receiving four intravenous doses of DISC-mGMCSF and the results from studies of neurovirulence, neuroinvasiveness, local tolerance in rabbit, general safety in mice and guinea pigs and safety in athymic nude mice were consistent with DISC being unable to replicate and cause disease. The vector could establish latency in local ganglia in mice, but at low efficiency, and could not reactivate infectious virions. Following intravenous administration, vector DNA was widely distributed up to Day 28, but by Day 56 had disappeared from gonads and brain and was only found in blood and liver. CONCLUSION: The panel of safety studies provided evidence that DISC-hGMCSF will be unable to replicate and cause disease, and has low toxicity in man. These data were presented to the Medicines Control Agency and the Gene Therapy Advisory Committee as part of the regulatory submissions for a clinical trial in melanoma patients. These submissions have been approved, and DISC-hGMCSF has now entered a phase I clinical trial in the UK by direct intratumoural injection.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Herpesvirus Humano 2/genética , Animais , Disponibilidade Biológica , Ensaios Clínicos Fase I como Assunto , DNA Viral/toxicidade , Vírus Defeituosos , Avaliação Pré-Clínica de Medicamentos , Feminino , Gânglios/virologia , Terapia Genética , Vetores Genéticos , Cobaias , Herpesvirus Humano 2/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase , Coelhos , Segurança , Latência Viral
10.
IUBMB Life ; 51(4): 201-5, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11569913

RESUMO

The sedentary habit of plants means that they must stand and fight environmental stresses that their mobile animal cousins can avoid. A range of these abiotic stresses initiate the production in plant cells of reactive oxygen and nitrogen species that ultimately lead to oxidative damage affecting the yield and quality of plant products. A complex network of enzyme systems, producing and quenching these reactive species operate in different organelles. It is the integration of these compartmented defense systems that coordinates an effective response to the various stresses. Future attempts to improve plant growth or yield must consider the complexity of inter-organelle signaling and protein targeting if they are to be successful in producing plants with resistance to a broad range of stresses. Here we highlight the role of pre-oxidant, antioxidant, and post-oxidant defense systems in plant mitochondria and the potential role of proteins targeted to both mitochondria and chloroplasts, in an integrated defense against oxidative damage in plants.


Assuntos
Mitocôndrias/metabolismo , Plantas/metabolismo , Animais , Antioxidantes/metabolismo , Modelos Biológicos , Oxidantes/metabolismo , Estresse Oxidativo , Proteínas de Plantas/metabolismo
11.
Cell Mol Life Sci ; 58(1): 61-71, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11229817

RESUMO

Biological nitrogen fixation involves the reduction of atmospheric N2 to ammonia by the bacterial enzyme nitrogenase. In legume-rhizobium symbioses, the nitrogenase-producing bacteria (bacteroids) are contained in the infected cells of root nodules within which they are enclosed by a plant membrane to form a structure known as the symbiosome. The plant provides reduced carbon to the bacteroids in exchange for fixed nitrogen, which is exported to the rest of the plant. This exchange is controlled by plant-synthesised transport proteins on the symbiosome membranes. This review summarises our current understanding of these transport processes, focusing on ammonia and amino acid transport.


Assuntos
Aminoácidos/metabolismo , Amônia/metabolismo , Membrana Celular/metabolismo , Fabaceae/metabolismo , Fabaceae/microbiologia , Fixação de Nitrogênio/fisiologia , Plantas Medicinais , Simbiose/fisiologia , Aminoácidos/biossíntese , Transporte Biológico , Proteínas de Transporte/metabolismo , Fabaceae/citologia , Nitrogênio/metabolismo , Nitrogenase/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Rhizobium/enzimologia , Rhizobium/metabolismo , Rhizobium/fisiologia
12.
J Bacteriol ; 182(10): 2838-44, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10781553

RESUMO

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing alpha-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of alpha-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate alpha-[U-(14)C]ketoglutarate and [U-(14)C]glutamate at rates similar to those of wild-type B. japonicum, indicating that there was an alternative route for alpha-ketoglutarate catabolism. Because cell extracts from B. japonicum decarboxylated [U-(14)C]glutamate very slowly, the gamma-aminobutyrate shunt is unlikely to be the pathway responsible for alpha-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent alpha-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP(i). Thin-layer chromatography showed that the product of alpha-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent alpha-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for alpha-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation.


Assuntos
Aldeído Oxirredutases/metabolismo , Bradyrhizobium/enzimologia , Carboxiliases/metabolismo , Ciclo do Ácido Cítrico , Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Bradyrhizobium/genética , Descarboxilação , Deleção de Genes , Glutamato Descarboxilase/metabolismo , Ácido Glutâmico/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Mutagênese , Fixação de Nitrogênio , Succinato-Semialdeído Desidrogenase
13.
Planta ; 209(1): 25-32, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10467028

RESUMO

The localization of H(+)-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H(+)-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H(+)-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids.


Assuntos
Glycine max/enzimologia , ATPases Translocadoras de Prótons/análise , ATPases Vacuolares Próton-Translocadoras , Microscopia Eletrônica , Raízes de Plantas/enzimologia , Raízes de Plantas/ultraestrutura , Glycine max/ultraestrutura
14.
FEBS Lett ; 454(3): 220-4, 1999 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-10431811

RESUMO

The alternative oxidase is a quinol oxidase of the respiratory chain of plants and some fungi and protists. Its activity is regulated by redox-sensitive disulphide bond formation between neighbouring subunits and direct interaction with certain alpha-ketoacids. To investigate these regulatory mechanisms, we undertook site-directed mutagenesis of soybean and Arabidopsis alternative oxidase cDNAs, and expressed them in tobacco plants and Escherichia coli, respectively. The homologous C99 and C127 residues of GmAOX3 and AtAOX1a, respectively, were changed to serine. In the plant system, this substitution prevented oxidative inactivation of alternative oxidase and rendered the protein insensitive to pyruvate activation, in agreement with the recent results from other laboratories [Rhoads et al. (1998) J. Biol. Chem. 273, 30750-30756; Vanlerberghe et al. (1998) Plant Cell 10, 1551-1560]. However, the mutated protein is instead activated specifically by succinate. Measurements of AtAOX1a activity in bacterial membranes lacking succinate dehydrogenase confirmed that the stimulation of the mutant protein's activity by succinate did not involve its metabolism. Examples of alternative oxidase proteins with the C to S substitution occur in nature and these oxidases are expected to be activated under most conditions in vivo, with implications for the efficiency of respiration in the tissues which express them.


Assuntos
Arabidopsis/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Oxirredutases/genética , Substituição de Aminoácidos , Arabidopsis/genética , Ativação Enzimática/genética , Escherichia coli/genética , Proteínas Mitocondriais , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Tóxicas , Nicotiana/genética
15.
FEBS Lett ; 447(1): 21-4, 1999 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-10218574

RESUMO

The alternative oxidase is found in the inner mitochondrial membranes of plants and some fungi and protists. A monoclonal antibody raised against the alternative oxidase from the aroid lily Sauromatum guttatum has been used extensively to detect the enzyme in these organisms. Using an immunoblotting strategy, the antibody binding site has been localised to the sequence RADEAHHRDVNH within the soybean alternative oxidase 2 protein. Examination of sequence variants showed that A2 and residues C-terminal to H7 are required for recognition by the monoclonal antibody raised against the alternative oxidase. The recognition sequence is highly conserved among all alternative oxidase proteins and is absolutely conserved in 12 of 14 higher plant sequences, suggesting that this antibody will continue to be extremely useful in studying the expression and synthesis of the alternative oxidase.


Assuntos
Especificidade de Anticorpos , Sequência Conservada , Glycine max/enzimologia , Oxirredutases/imunologia , Proteínas de Plantas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Sítios de Ligação , Proteínas Mitocondriais , Dados de Sequência Molecular , Oxirredutases/genética , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/imunologia , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
16.
J Endocrinol ; 157(3): 361-71, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9691970

RESUMO

Expression of a gene can be controlled at many levels, including transcription, mRNA splicing, mRNA stability, translation and post-translational events such as protein stability and modification. The majority of studies to date have focused on transcriptional control mechanisms, but the importance of post-transcriptional mechanisms in regulating gene expression in eukaryotes is becoming increasingly clear. In this short review, selected examples of post-transcriptional gene regulatory mechanisms operating in both lower and higher eukaryotes will be used to highlight the plethora of such mechanisms already identified. The underlying theme is that post-transcriptional gene regulation relies on specific RNA-protein interactions that either result in the targeted degradation of the mRNA or prevent access of the ribosome to the translation start codon. Such interactions can occur in the 5' or 3' untranslated regions of an mRNA or within the decoded portion of the molecule. The importance of these regulatory mechanisms in a range of biological systems is also illustrated.


Assuntos
Regulação da Expressão Gênica , Mamíferos/genética , Saccharomyces cerevisiae/genética , Animais , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Transcrição Gênica
17.
Science ; 281(5380): 1202-6, 1998 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-9712587

RESUMO

Nitrogen-fixing bacteroids in legume root nodules are surrounded by the plant-derived peribacteroid membrane, which controls nutrient transfer between the symbionts. A nodule complementary DNA (GmSAT1) encoding an ammonium transporter has been isolated from soybean. GmSAT1 is preferentially transcribed in nodules and immunoblotting indicates that GmSAT1 is located on the peribacteroid membrane. [14C]methylammonium uptake and patch-clamp analysis of yeast expressing GmSAT1 demonstrated that it shares properties with a soybean peribacteroid membrane NH4+ channel described elsewhere. GmSAT1 is likely to be involved in the transfer of fixed nitrogen from the bacteroid to the host.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Glycine max/genética , Compostos de Amônio Quaternário/metabolismo , Proteínas de Soja , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Proteínas de Transporte/química , Membrana Celular/metabolismo , DNA Complementar , Canais Iônicos/metabolismo , Cinética , Metilaminas/metabolismo , Dados de Sequência Molecular , Organelas/metabolismo , Técnicas de Patch-Clamp , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Potássio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Glycine max/química , Glycine max/metabolismo , Glycine max/microbiologia , Esferoplastos/metabolismo , Simbiose , Transformação Genética
18.
Plant Mol Biol ; 36(1): 171-81, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9484472

RESUMO

Nuclear-encoded mitochondrial precursor proteins are proteolytically processed inside the mitochondrion after import. The general mitochondrial processing activity in plant mitochondria has been shown to be integrated into the cytochrome bc1 complex of the respiratory chain. Here we investigate the occurrence of an additional, matrix-located processing activity by incubation of the precursors of the soybean mitochondrial proteins, alternative oxidase, the FAd subunit of the ATP synthetase and the tobacco F1 beta subunit of the ATP synthase, with the membrane and soluble components of mitochondria isolated from soybean cotyledons and spinach leaves. A matrix-located peptidase specifically processed the precursors to the predicted mature form in a reaction which was sensitive to orthophenanthroline, a characteristic inhibitor of mitochondrial processing peptidase (MPP). The specificity of the matrix peptidase was illustrated by the inhibition of processing of the alternative oxidase precursor in both soybean and spinach matrix extracts upon altering a single amino acid residue in the targeting presequence (-2 Arg to Gly). Additionally, there was no evidence for general proteolysis of precursor proteins incubated with the matrix. The purity of the matrix fractions was ascertained by spectrophotometric and immunological analyses. The results demonstrate that there is a specific processing activity in the matrix of soybean and spinach in addition to the previously well characterized membrane-bound MPP integrated into the cytochrome bcl complex of the respiratory chain.


Assuntos
Endopeptidases/metabolismo , Glycine max/enzimologia , Mitocôndrias/enzimologia , Processamento de Proteína Pós-Traducional , Núcleo Celular/metabolismo , Cotilédone , Endopeptidases/isolamento & purificação , Proteínas Mitocondriais , Mutagênese Sítio-Dirigida , Oxirredutases/biossíntese , Folhas de Planta , Proteínas de Plantas/metabolismo , Plantas Tóxicas , Precursores de Proteínas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Spinacia oleracea/enzimologia , Nicotiana/enzimologia
19.
Plant Physiol ; 114(2): 455-66, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9193084

RESUMO

The alternative oxidase (AOX) of the soybean (Glycine max L.) inner mitochondrial membrane is encoded by a multigene family (Aox) with three known members. Here, the Aox2 and Aox3 primary translation products, deduced for cDNA analysis, were found to be 38.1 and 36.4 Kd, respectively. Direct N-terminal sequencing of partially purified AOX from cotyledons demonstrates that the mature proteins are 31.8 and 31.6 KD, respectively, implying that processing occurs upon import of these proteins into the mitochondrion. Sequence comparisons show that the processing of plant AOX proteins occurs at a characteristic site and that the AOX2 and AOX3 proteins are more similar to one another than to other AOX proteins, including soybean AOX1. Transcript analysis using a polymerase chain reaction-based assay in conjunction with immunoblot experiments indicates that soybean Aox genes are differentially expressed in a tissue-dependent manner. Moreover, the relative abundance of both Aox2 transcripts and protein in cotyledons increase upon greening of dark-grown seedlings. These results comprehensively explain the multiple AOX-banding patterns observed on immunoblots of mitochondrial proteins isolated from various soybean tissues by matching protein bands with gene products.


Assuntos
Genes de Plantas , Glycine max/genética , Mitocôndrias/genética , Família Multigênica , Oxirredutases/genética , Sequência de Aminoácidos , Clonagem Molecular , Sequência Conservada , Cotilédone/enzimologia , DNA Complementar/genética , Expressão Gênica , Luz , Mitocôndrias/enzimologia , Proteínas Mitocondriais , Dados de Sequência Molecular , Oxirredutases/biossíntese , Proteínas de Plantas , Reação em Cadeia da Polimerase , Conformação Proteica , Sinais Direcionadores de Proteínas , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Glycine max/enzimologia , Glycine max/efeitos da radiação , Especificidade da Espécie , Distribuição Tecidual , Ubiquinona/análise
20.
Percept Mot Skills ; 84(3 Pt 2): 1135-8, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9229425

RESUMO

The Group Embedded Figures Test was administered to 63 undergraduates in a program for a Bachelor of Applied Arts in Information management. Distribution characteristics, sex differences, reliability, and internal consistency measures for this sample were compared with those for Witkin's original sample. In addition, item difficulty and discrimination coefficients are provided. Scores for this group show desirable measurement characteristics.


Assuntos
Área de Dependência-Independência , Percepção de Forma , Testes Psicológicos , Adulto , Cognição , Feminino , Humanos , Masculino , Personalidade , Testes Psicológicos/estatística & dados numéricos , Psicometria , Reprodutibilidade dos Testes , Distribuição por Sexo , Fatores Sexuais
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