RESUMO
Recently, we reported estimates of anaplerotic carbon flux through the oxidative pentose phosphate pathway (OPPP) in chloroplasts into the Calvin-Benson cycle. These estimates were based on intramolecular hydrogen isotope analysis of sunflower leaf starch. However, the isotope method is believed to underestimate the actual flux at low atmospheric CO2 concentration (Ca ). Since the OPPP releases CO2 and reduces NADP+ , it can be expected to affect leaf gas exchange under both rubisco- and RuBP-regeneration-limited conditions. Therefore, we expanded Farquhar-von Caemmerer-Berry models to account for OPPP metabolism. Based on model parameterisation with values from the literature, we estimated OPPP-related effects on leaf carbon and energy metabolism in the sunflowers analysed previously. We found that flux through the plastidial OPPP increases both above and below Ca ≈ 450 ppm (the condition the plants were acclimated to). This is qualitatively consistent with our previous isotope-based estimates, yet gas-exchange-based estimates are larger at low Ca . We discuss our results in relation to regulatory properties of the plastidial and cytosolic OPPP, the proposed variability of CO2 mesophyll conductance, and the contribution of day respiration to the A/Ci curve drop at high Ca . Furthermore, we critically examine the models and parameterisation and derive recommendations for follow-up studies.
Assuntos
Dióxido de Carbono , Via de Pentose Fosfato , Dióxido de Carbono/metabolismo , Fotossíntese , Folhas de Planta/metabolismo , Respiração , Estresse OxidativoRESUMO
As the central carbon uptake pathway in photosynthetic cells, the Calvin-Benson cycle is among the most important biochemical cycles for life on Earth. A carbon flux of anaplerotic origin (i.e. through the chloroplast-localized oxidative branch of the pentose phosphate pathway) into the Calvin-Benson cycle was proposed recently. Here, we measured intramolecular deuterium abundances in leaf starch of Helianthus annuus grown at varying ambient CO2 concentrations, Ca . Additionally, we modelled deuterium fractionations expected for the anaplerotic pathway and compared modelled with measured fractionations. We report deuterium fractionation signals at H1 and H2 of starch glucose. Below a Ca change point, these signals increase with decreasing Ca consistent with modelled fractionations by anaplerotic flux. Under standard conditions (Ca = 450 ppm corresponding to intercellular CO2 concentrations, Ci , of 328 ppm), we estimate negligible anaplerotic flux. At Ca = 180 ppm (Ci = 140 ppm), more than 10% of the glucose-6-phosphate entering the starch biosynthesis pathway is diverted into the anaplerotic pathway. In conclusion, we report evidence consistent with anaplerotic carbon flux into the Calvin-Benson cycle in vivo. We propose the flux may help to: maintain high levels of ribulose 1,5-bisphosphate under source-limited growth conditions to facilitate photorespiratory nitrogen assimilation required to build-up source strength; and counteract oxidative stress.
Assuntos
Hidrogênio , Fotossíntese , Ciclo do Carbono , Isótopos , Via de Pentose FosfatoRESUMO
Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences. To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961-1995). We found fractionation signals (i.e. temporal variability in deuterium abundance) at glucose H1 and H2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain ≈ 60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth. We propose the signals are introduced at the leaf level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.
Assuntos
Pinus , Árvores , Isótopos de Carbono/metabolismo , Glucose/metabolismo , Hidrogênio , Isótopos de Oxigênio/metabolismo , Pinus/metabolismoRESUMO
Stable isotopes at natural abundance are key tools to study physiological processes occurring outside the temporal scope of manipulation and monitoring experiments. Whole-molecule carbon isotope ratios (13C/12C) enable assessments of plant carbon uptake yet conceal information about carbon allocation. Here, we identify an intramolecular 13C/12C signal at tree-ring glucose C-5 and C-6 and develop experimentally testable theories on its origin. More specifically, we assess the potential of processes within C3 metabolism for signal introduction based (inter alia) on constraints on signal propagation posed by metabolic networks. We propose that the intramolecular signal reports carbon allocation into major metabolic pathways in actively photosynthesizing leaf cells including the anaplerotic, shikimate, and non-mevalonate pathway. We support our theoretical framework by linking it to previously reported whole-molecule 13C/12C increases in cellulose of ozone-treated Betula pendula and a highly significant relationship between the intramolecular signal and tropospheric ozone concentration. Our theory postulates a pronounced preference for leaf cytosolic triose-phosphate isomerase to catalyse the forward reaction in vivo (dihydroxyacetone phosphate to glyceraldehyde 3-phosphate). In conclusion, intramolecular 13C/12C analysis resolves information about carbon uptake and allocation enabling more comprehensive assessments of carbon metabolism than whole-molecule 13C/12C analysis.
Assuntos
Plantas , Árvores , Carbono/metabolismo , Isótopos de Carbono/metabolismo , Glucose/metabolismo , Folhas de Planta/metabolismo , Plantas/metabolismo , Árvores/metabolismoRESUMO
Peatlands store carbon in the form of dead organic residues. Climate change and human impact impose risks on the sustainability of the peatlands carbon balance due to increased peat decomposition. Here, we investigated molecular changes in the upper peat layers (0-40 cm), inferred from high-resolution vertical depth profiles, from a boreal peatland using two-dimensional 1H-13C nuclear magnetic resonance (NMR) spectroscopy, and comparison to δ13C, δ15N, and carbon and nitrogen content. Effects of hydrological conditions were investigated at respective sites: natural moist, drainage ditch, and natural dry. The molecular characterization revealed preferential degradation of specific side-chain linkages of xylan-type hemicelluloses within 0-14 cm at all sites, indicating organic matter losses up to 25%. In contrast, the xylan backbone, galactomannan-type hemicelluloses, and cellulose were more resistant to degradation and accumulated at the natural moist and drainage site. δ13C, δ15N, and carbon and nitrogen content did not correlate with specific hemicellulose structures but reflected changes in total carbohydrates. Our analysis provides novel insights into peat carbohydrate decomposition and indicates substantial organic matter losses in the acrotelm due to the degradation of specific hemicellulose structures. This suggests that variations in hemicellulose content and structure influence peat stability, which may have important implications with respect to climate change.
Assuntos
Solo , Xilanos , Humanos , Solo/química , Carbono/química , Nitrogênio/análiseRESUMO
Sphagnum mosses account for most accumulated dead organic matter in peatlands. Therefore, understanding their responses to increasing atmospheric CO2 is needed for estimating peatland C balances under climate change. A key process is photorespiration: a major determinant of net photosynthetic C assimilation that depends on the CO2 to O2 ratio. We used climate chambers to investigate photorespiratory responses of Sphagnum fuscum hummocks to recent increases in atmospheric CO2 (from 280 to 400 ppm) under different water table, temperature, and light intensity levels. We tested the photorespiratory variability using a novel method based on deuterium isotopomers (D6S /D6R ratio) of photosynthetic glucose. The effect of elevated CO2 on photorespiration was highly dependent on water table. At low water table (-20 cm), elevated CO2 suppressed photorespiration relative to C assimilation, thus substantially increasing the net primary production potential. In contrast, a high water table (~0 cm) favored photorespiration and abolished this CO2 effect. The response was further tested for Sphagnum majus lawns at typical water table levels (~0 and -7 cm), revealing no effect of CO2 under those conditions. Our results indicate that hummocks, which typically experience low water table levels, benefit from the 20th century's increase in atmospheric CO2 .
Assuntos
Dióxido de Carbono/metabolismo , Sphagnopsida/fisiologia , Biomassa , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Cloroplastos/metabolismo , Água Subterrânea , Luz , Isótopos de Nitrogênio/análise , Isótopos de Nitrogênio/metabolismo , Fotossíntese , Suécia , Temperatura , Água/análise , Água/metabolismoRESUMO
Within the plant and Earth sciences, stable isotope analysis is a versatile tool conveying information (inter alia) about plant physiological and paleoclimate variability across scales. Here, we identify a 13C signal (i.e. systematic 13C/12C variation) at tree-ring glucose C-4 and report an experimentally testable theory on its origin. We propose the signal is introduced by glyceraldehyde-3-phosphate dehydrogenases in the cytosol of leaves. It conveys two kinds of (potentially convoluted) information: (i) commitment of glyceraldehyde 3-phosphate to 3-phosphoglycerate versus fructose 1,6-bisphosphate metabolism; and (ii) the contribution of non-phosphorylating versus phosphorylating glyceraldehyde-3-phosphate dehydrogenase to catalysing the glyceraldehyde 3-phosphate to 3-phosphoglycerate forward reaction of glycolysis. The theory is supported by 13C fractionation modelling. Modelling results provide the first evidence in support of the cytosolic oxidation-reduction (COR) cycle, a carbon-neutral mechanism supplying NADPH at the expense of ATP and NADH, which may help to maintain leaf-cytosolic redox balances. In line with expectations related to COR cycling, we found a positive correlation between air vapour pressure deficit and 13C discrimination at glucose C-4. Overall, 13C-4 signal analysis may enable an improved understanding of leaf carbon and energy metabolism.
Assuntos
Glucose , Gliceraldeído-3-Fosfato Desidrogenases , Ciclo do Carbono , Isótopos de Carbono/metabolismo , Citosol/metabolismo , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicólise , Cinética , Folhas de Planta/metabolismoRESUMO
Hydrogen (H) isotope ratio (δ2 H) analyses of plant organic compounds have been applied to assess ecohydrological processes in the environment despite a large part of the δ2 H variability observed in plant compounds not being fully elucidated. We present a conceptual biochemical model based on empirical H isotope data that we generated in two complementary experiments that clarifies a large part of the unexplained variability in the δ2 H values of plant organic compounds. The experiments demonstrate that information recorded in the δ2 H values of plant organic compounds goes beyond hydrological signals and can also contain important information on the carbon and energy metabolism of plants. Our model explains where 2 H-fractionations occur in the biosynthesis of plant organic compounds and how these 2 H-fractionations are tightly coupled to a plant's carbon and energy metabolism. Our model also provides a mechanistic basis to introduce H isotopes in plant organic compounds as a new metabolic proxy for the carbon and energy metabolism of plants and ecosystems. Such a new metabolic proxy has the potential to be applied in a broad range of disciplines, including plant and ecosystem physiology, biogeochemistry and palaeoecology.
Assuntos
Carboidratos/biossíntese , Fracionamento Químico/métodos , Deutério/metabolismo , Lipídeos/biossíntese , Compostos Orgânicos/metabolismo , Plantas/metabolismo , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Respiração Celular , Hidrogênio/metabolismo , Fotossíntese , Folhas de Planta/metabolismoRESUMO
Terrestrial vegetation currently absorbs approximately a third of anthropogenic CO2 emissions, mitigating the rise of atmospheric CO2. However, terrestrial net primary production is highly sensitive to atmospheric CO2 levels and associated climatic changes. In C3 plants, which dominate terrestrial vegetation, net photosynthesis depends on the ratio between photorespiration and gross photosynthesis. This metabolic flux ratio depends strongly on CO2 levels, but changes in this ratio over the past CO2 rise have not been analyzed experimentally. Combining CO2 manipulation experiments and deuterium NMR, we first establish that the intramolecular deuterium distribution (deuterium isotopomers) of photosynthetic C3 glucose contains a signal of the photorespiration/photosynthesis ratio. By tracing this isotopomer signal in herbarium samples of natural C3 vascular plant species, crops, and a Sphagnum moss species, we detect a consistent reduction in the photorespiration/photosynthesis ratio in response to the â¼100-ppm CO2 increase between â¼1900 and 2013. No difference was detected in the isotopomer trends between beet sugar samples covering the 20th century and CO2 manipulation experiments, suggesting that photosynthetic metabolism in sugar beet has not acclimated to increasing CO2 over >100 y. This provides observational evidence that the reduction of the photorespiration/photosynthesis ratio was ca. 25%. The Sphagnum results are consistent with the observed positive correlations between peat accumulation rates and photosynthetic rates over the Northern Hemisphere. Our results establish that isotopomers of plant archives contain metabolic information covering centuries. Our data provide direct quantitative information on the "CO2 fertilization" effect over decades, thus addressing a major uncertainty in Earth system models.
Assuntos
Dióxido de Carbono/metabolismo , Fotossíntese , Plantas/metabolismo , Isótopos de Carbono , DeutérioRESUMO
Mitigation of N-nitrosodimethylamine (NDMA) and other hazardous water disinfection byproducts (DBP) is currently hampered by a limited understanding of DBP formation mechanisms. Because variations of the stable isotope composition of NDMA can potentially reveal reaction pathways and precursor compounds, we developed a method for the compound-specific isotope analysis (CSIA) of (13)C/(12)C, (15)N/(14)N, and (2)H/(1)H ratios of NDMA by gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). Method quantification limits for the accurate isotope analysis of NDMA, N-nitrosodiethyl-, -dipropyl-, and -dibutylamine as well as N-nitrosopyrrolidine were between 0.18 to 0.60 nmol C, 0.40 to 0.80 nmol N, and 2.2 to 5.8 nmol H injected on column. Coupling solid phase extraction (SPE) to GC/IRMS enabled the precise quantification of C, N, and H isotope ratios of NDMA in aqueous samples at concentrations of 0.6 µM (45 µg L(-1)). We validated the proposed method with a laboratory experiment, in which NDMA was formed with stoichiometric yield (97 ± 4%) through chloramination of the pharmaceutical ranitidine (3 µM). δ(13)C and δ(2)H values of NDMA remained constant during NDMA formation while its δ(15)N increased due to a reaction at a N atom in the rate-limiting step of NDMA formation. The δ(2)H value of NDMA determined by SPE-GC/IRMS also corresponded well to the δ(2)H value of the N(CH3)2-group of ranitidine measured by quantitative deuterium nuclear magnetic resonance spectroscopy. This observation implies that the N(CH3)2-moiety of ranitidine is transferred to NDMA without being chemically altered and illustrates the accuracy of the proposed method.
Assuntos
Isótopos de Carbono/química , Dimetilnitrosamina/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Hidrogênio/química , Isótopos de Nitrogênio/química , Poluentes Químicos da Água/análise , Água/química , Desinfecção , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Extração em Fase SólidaRESUMO
Understanding the composition of organic phosphorus (P) in soils is relevant to various disciplines, from agricultural sciences to ecology. Despite past efforts, the precise nature of soil organic P remains an enigma, especially that of the orthophosphate monoesters, which dominate 31P NMR spectra of NaOH-EDTA extracts of soils worldwide. The monoester region often exhibits an unidentified, broad background believed to represent high molecular weight (MW) P. We investigated this monoester background using 1D 31P NMR and 2D 1H31P NMR, as well as 31P transverse relaxation (T2) measurements to calculate its intrinsic linewidth and relate it to MW. Analyzing seven soils from different ecosystems, we observed linewidths of 0.5 to 3 Hz for resolved monoester signals and the background, indicating that it consists of many, possibly >100, sharp signals associated with small (<1.5 kDa) organic P molecules. This result was further supported by 2D 1H31P NMR spectra revealing signals not resolved in the 1D spectra. Our findings align with 31P NMR studies detecting background signals in soil-free samples and modern evidence that alkali-soluble soil organic matter consists of self-assemblies of small organic compounds mimicking large molecules.
RESUMO
The global soil carbon pool is approximately three times larger than the contemporary atmospheric pool, therefore even minor changes to its integrity may have major implications for atmospheric CO2 concentrations. While theory predicts that the chemical composition of organic matter should constitute a master control on the temperature response of its decomposition, this relationship has not yet been fully demonstrated. We used laboratory incubations of forest soil organic matter (SOM) and fresh litter material together with NMR spectroscopy to make this connection between organic chemical composition and temperature sensitivity of decomposition. Temperature response of decomposition in both fresh litter and SOM was directly related to the chemical composition of the constituent organic matter, explaining 90% and 70% of the variance in Q10 in litter and SOM, respectively. The Q10 of litter decreased with increasing proportions of aromatic and O-aromatic compounds, and increased with increased contents of alkyl- and O-alkyl carbons. In contrast, in SOM, decomposition was affected only by carbonyl compounds. To reveal why a certain group of organic chemical compounds affected the temperature sensitivity of organic matter decomposition in litter and SOM, a more detailed characterization of the (13) C aromatic region using Heteronuclear Single Quantum Coherence (HSQC) was conducted. The results revealed considerable differences in the aromatic region between litter and SOM. This suggests that the correlation between chemical composition of organic matter and the temperature response of decomposition differed between litter and SOM. The temperature response of soil decomposition processes can thus be described by the chemical composition of its constituent organic matter, this paves the way for improved ecosystem modeling of biosphere feedbacks under a changing climate.
Assuntos
Dióxido de Carbono/metabolismo , Ecossistema , Substâncias Húmicas , Solo/química , Temperatura , Mudança Climática , Espectroscopia de Ressonância Magnética , SuéciaRESUMO
A large proportion of the global soil carbon pool is stored in soils of high-latitude ecosystems in which microbial processes and production of greenhouse gases proceed during the winter months. It has been suggested that microorganisms have limited ability to sequester substrates at temperatures around and below 0 °C and that a metabolic shift to dominance of catabolic processes occurs around these temperatures. However, there are contrary indications that anabolic processes can proceed, because microbial growth has been observed at far lower temperatures. Therefore, we investigated the utilization of the microbial substrate under unfrozen and frozen conditions in a boreal forest soil across a temperature range from -9 °C to +9 °C, by using gas chromatography-isotopic ratio mass spectrometry and (13)C magic-angle spinning NMR spectroscopy to determine microbial turnover and incorporation of (13)C-labeled glucose. Our results conclusively demonstrate that the soil microorganisms maintain both catabolic (CO(2) production) and anabolic (biomass synthesis) processes under frozen conditions and that no significant differences in carbon allocation from [(13)C]glucose into [(13)C]CO(2) and cell organic (13)C-compounds occurred between +9 °C and -4 °C. The only significant metabolic changes detected were increased fluidity of the cell membranes synthesized at frozen conditions and increased production of glycerol in the frozen samples. The finding that the processes in frozen soil are similar to those in unfrozen soil has important implications for our general understanding and conceptualization of soil carbon dynamics in high-latitude ecosystems.
Assuntos
Aclimatação , Congelamento , Processos Heterotróficos , Microbiologia do Solo , Carbono/metabolismo , Temperatura Baixa , Glucose/metabolismo , Glicerol/análise , Espectroscopia de Ressonância Magnética , Estações do Ano , Solo , Árvores/microbiologiaRESUMO
Organic phosphorus (P) compounds represent a major component of soil P in many soils and are key sources of P for microbes and plants. Solution NMR (nuclear magnetic resonance spectroscopy) is a powerful technique for characterizing organic P species. However, (31)P NMR spectra are often complicated by overlapping peaks, which hampers identification and quantification of the numerous P species present in soils. Overlap is often exacerbated by the presence of paramagnetic metal ions, even if they are in complexes with EDTA following NaOH/EDTA extraction. By removing paramagnetic impurities using a new precipitation protocol, we achieved a dramatic improvement in spectral resolution. Furthermore, the obtained reduction in line widths enabled the use of multidimensional NMR methods to resolve overlapping (31)P signals. Using the new protocol on samples from two boreal humus soils with different Fe contents, 2D (1)H-(31)P correlation spectra allowed unambiguous identification of a large number of P species based on their (31)P and (1)H chemical shifts and their characteristic coupling patterns, which would not have been possible using previous protocols. This approach can be used to identify organic P species in samples from both terrestrial and aquatic environments increasing our understanding of organic P biogeochemistry.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Compostos Orgânicos/análise , Fósforo/análise , Prótons , Solo/química , Ácido Edético/química , Isótopos de Fósforo , Hidróxido de Sódio/químicaRESUMO
Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigour of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and nuclear magnetic resonance. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle-host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.
Assuntos
Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Helicobacter pylori/patogenicidade , Fatores de Virulência/isolamento & purificação , Adesinas Bacterianas/metabolismo , Antígenos de Bactérias/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Mucosa Gástrica/microbiologia , Humanos , Fosfolipídeos/análise , Técnicas de Cultura de TecidosRESUMO
Natural peatlands contribute significantly to global carbon sequestration and storage of biomass, most of which derives from Sphagnum peat mosses. Atmospheric CO2 levels have increased dramatically during the twentieth century, from 280 to > 400 ppm, which has affected plant carbon dynamics. Net carbon assimilation is strongly reduced by photorespiration, a process that depends on the CO2 to O2 ratio. Here we investigate the response of the photorespiration to photosynthesis ratio in Sphagnum mosses to recent CO2 increases by comparing deuterium isotopomers of historical and contemporary Sphagnum tissues collected from 36 peat cores from five continents. Rising CO2 levels generally suppressed photorespiration relative to photosynthesis but the magnitude of suppression depended on the current water table depth. By estimating the changes in water table depth, temperature, and precipitation during the twentieth century, we excluded potential effects of these climate parameters on the observed isotopomer responses. Further, we showed that the photorespiration to photosynthesis ratio varied between Sphagnum subgenera, indicating differences in their photosynthetic capacity. The global suppression of photorespiration in Sphagnum suggests an increased net primary production potential in response to the ongoing rise in atmospheric CO2, in particular for mire structures with intermediate water table depths.
RESUMO
Recent evidence supports the idea that early aggregates, protein, and lipoprotein oligomers but not large aggregates like fibrils that are formed at late stages of the aggregation process are responsible for cytotoxicity. Oligomers can interact with the cellular plasma membrane affecting its structure and/or dynamics or may be taken up by the cells. In either case, disparate cascades of molecular interactions are activated in the attempt to counteract the disturbance induced by the oligomers. If unsuccessful, cell death follows. Here, we study the molecular and cellular mechanisms underlying PC12 cell death caused by ELOA oligomers. ELOA, a lipoprotein complex formed by equine lysozyme (EL) and oleic acid (OA), induces cell death in all tested cell lines, but the actual mechanism of its action is not known. We have used methods with single-molecule sensitivity, fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and confocal laser scanning microscopy (CLSM) imaging by avalanche photodiodes (APD), so-called APD imaging, to study ELOA interactions with the plasma membrane in live PC12 cells. We detected ELOA accumulation in the cell surroundings, observed ELOA interactions with the plasma membrane, and local changes in plasma membrane lipid dynamics in the vicinity of ELOA complexes. These interactions resulted in plasma membrane rupture, followed by rapid influx and distribution of ELOA inside the already dead cell. In order to probe the ELOA-plasma membrane interaction sites at the molecular and atomic levels, the ELOA complexes were further studied by photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, nuclear magnetic resonance (NMR) and atomic force microscopy (AFM). We observed a novel mechanism of oligomer toxicity-cell death induced by continuous disturbance of the plasma membrane, eventually causing permanent plasma membrane damage and identified the sites in ELOA that are potentially involved in the interactions with the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Lipoproteínas/metabolismo , Muramidase/metabolismo , Ácido Oleico/metabolismo , Animais , Sítios de Ligação , Sobrevivência Celular , Cor , Corantes Fluorescentes/metabolismo , Cavalos , Células PC12 , Ligação Proteica , Ratos , Espectrometria de FluorescênciaRESUMO
Using (13)C spin relaxation NMR in combination with molecular dynamic (MD) simulations, we characterized internal motions within double-stranded DNA on the pico- to nano-second time scale. We found that the C-H vectors in all cytosine ribose moieties within the Dickerson-Drew dodecamer (5'-CGCGAATTCGCG-3') are subject to high amplitude motions, while the other nucleotides are essentially rigid. MD simulations showed that repuckering is a likely motional model for the cytosine ribose moiety. Repuckering occurs with a time constant of around 100 ps. Knowledge of DNA dynamics will contribute to our understanding of the recognition specificity of DNA-binding proteins such as cytosine methyltransferase.
Assuntos
Citosina/química , DNA/química , Ribose/química , Simulação por Computador , Movimento (Física) , Ressonância Magnética Nuclear BiomolecularRESUMO
Lipids play a central role in numerous biological events, ranging from normal physiological processes to host-pathogen interactions. The proposed semiconstant-time (31)P,(1)H-COSY NMR experiment provides identification of known and structural characterization of unknown phospholipids in complex membrane extracts with high sensitivity, based on the combination of their (1)H and (31)P chemical shifts and coupling patterns. Furthermore, the spectra allow quantification of phospholipid composition. Analysis of the phospholipid composition of Helicobacter pylori, the causative agent of peptic ulcer disease, showed the presence of uncommon phospholipids. This novel NMR approach allows the study of changes in membrane composition in response to biological stimuli and opens up the possibility of identifying soluble phosphorus species in a number of research fields.
Assuntos
Helicobacter pylori/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fosfolipídeos/análise , Cardiolipinas/análise , Cardiolipinas/química , Membrana Celular/química , Fosfatidilcolinas/análise , Fosfatidilcolinas/química , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/química , Fosfatidilgliceróis/análise , Fosfatidilgliceróis/química , Fosfolipídeos/químicaRESUMO
The number of regulatory RNAs with identified non-canonical structures is increasing, and structural transitions often play a role in their biological function. This stimulates interest in internal motions of RNA, which can underlie structural transitions. Heteronuclear NMR relaxation measurements, which are commonly used to study internal motion, only report on local motions of few sites within the molecule. Here we have studied a 27-nt segment of the human hepatitis B virus (HBV) pregenomic RNA, which is essential for viral replication. We combined heteronuclear relaxation with the new off-resonance ROESY technique, which reports on internal motions of H,H contacts. Using off-resonance ROESY, we could for the first time detect motion of through-space H,H contacts, such as in intra-residue base-ribose contacts or inter-nucleotide contacts, both essential for NMR structure determination. Motions in non-canonical structure elements were found primarily on the sub-nanosecond timescale. Different patterns of mobility were observed among several mobile nucleotides. The most mobile nucleotides are highly conserved among different HBV strains, suggesting that their mobility patterns may be necessary for the RNA's biological function.