934 resultados para Reconstituted glaciers
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Understanding abrupt climate changes requires detailed spatial/temporal records of such changes, and to make these records, we need rapidly responding, geographically widespread climate trackers. Glacial systems are such trackers, and recent additions to the stratigraphic record show overall synchronous response of glacial systems to climate change reflecting global atmosphere conditions.
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In this study the interplay of mitochondria and peroxisomes in photorespiration was simulated in a reconstituted system of isolated mitochondria and peroxisomes from spinach (Spinacia oleracea L.) leaves. The mitochondria oxidizing glycine produced serine, which was reduced in the peroxisomes to glycerate. The required reducing equivalents were provided by the mitochondria via the malate-oxaloacetate (OAA) shuttle, in which OAA was reduced in the mitochondrial matrix by NADH generated during glycine oxidation. The rate of peroxisomal glycerate formation, as compared with peroxisomal protein, resembled the corresponding rate required during leaf photosynthesis under ambient conditions. When the reconstituted system produced glycerate at this rate, the malate-to-OAA ratio was in equilibrium with a ratio of NADH/NAD of 8.8 × 10−3. This low ratio is in the same range as the ratio of NADH/NAD in the cytosol of mesophyll cells of intact illuminated spinach leaves, as we had estimated earlier. This result demonstrates that in the photorespiratory cycle a transfer of redox equivalents from the mitochondria to peroxisomes, as postulated from separate experiments with isolated mitochondria and peroxisomes, can indeed operate under conditions of the very low reductive state of the NADH/NAD system prevailing in the cytosol of mesophyll cells in a leaf during photosynthesis.
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We created a "knockout" embryonic stem cell via targeted disruption of the phosphatidylinositol glycan class A (Pig-a) gene, resulting in loss of expression of cell surface glycosyl phosphatidylinositol-anchored proteins and reproducing the mutant phenotype of the human disease paroxysmal nocturnal hemoglobinuria. Morphogenesis of Pig-a- embryoid bodies (EB) in vitro was grossly aberrant and, unlike EB derived from normal embryonic stem cells, Pig-A EB produced no secondary hematopoietic colonies. Chimeric EB composed of control plus Pig-A- cells, however, appeared normal, and hematopoiesis from knock-out cells was reconstituted. Transfer in situ of glycosyl phosphatidylinositol-anchored proteins from normal to knock-out cells was demonstrated by two-color fluorescent analysis, suggesting a possible mechanism for these functional effects. Hematopoietic cells with mutated PIG-A genes in humans with paroxysmal nocturnal hemoglobinuria may be subject to comparable pathophysiologic processes and amenable to similar therapeutic protein transfer.
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The electrophoretic export of ATP against the import of ADP in mitochondria bridges the intra- versus extramitochondrial ATP potential gap. Here we report that the electrical nature of the ADP/ATP exchange by the mitochondrial ADP/ATP carrier (AAC) can be directly studied by measuring the electrical currents via capacitive coupling of AAC-containing vesicles on a planar lipid membrane. The currents were induced by the rapid liberation of ATP or ADP with UV flash photolysis from caged nucleotides. Six different transport modes of the AAC were studied: heteroexchange with either ADP or ATP inside the vesicles, initiated by photolysis of caged ATP or ADP; homoexchange with ADPex/ADPin or ATPex/ATPin; and caged ADP or ATP with unloaded vesicles. The heteroexchange produced the largest currents with the longest duration in line with the electrical charge difference ATP4- versus ADP3-. Surprisingly, also in the homoexchange and with unloaded vesicles, small currents were measured with shorter duration. In all three modes with caged ATP, a negative charge moved into the vesicles and with caged ADP it moved out of the vesicles. All currents were completely inhibited by a mixture of the inhibitors of the AAC, carboxyatractyloside and hongkrekate, which proves that the currents are exclusively due to AAC function. The observed charge movements in the heteroexchange system agree with the prediction from transport studies in mitochondria and reconstituted vesicles. The unexpected charge movements in the homoexchange or unloaded systems are interpreted to reveal transmembrane rearrangements of charged sites in the AAC when occupied with ADP or ATP. The results also indicate that not only ATP4- but also ADP3- contribute, albeit in opposite direction, to the electrical nature of the ADP/ATP exchange, which is at variance with former conclusions from biochemical transport studies. These measurements open up new avenues of studying the electrical interactions of ADP and ATP with the AAC.
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Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.
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We present a simple, rapid procedure for reconstitution of Escherichia coli RNA polymerase holoenzyme (RNAP) from individual recombinant alpha, beta, beta', and sigma 70 subunits. Hexahistidine-tagged recombinant alpha subunit purified by batch-mode metal-ion-affinity chromatography is incubated with crude recombinant beta, beta', and sigma 70 subunits from inclusion bodies, and the resulting reconstituted recombinant RNAP is purified by batch-mode metal-ion-affinity chromatography. RNAP prepared by this procedure is indistinguishable from RNAP prepared by conventional methods with respect to subunit stoichiometry, alpha-DNA interaction, catabolite gene activator protein (CAP)-independent transcription, and CAP-dependent transcription. Experiments with alpha (1-235), an alpha subunit C-terminal deletion mutant, establish that the procedure is suitable for biochemical screening of subunit lethal mutants.
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Substantial retreat or disintegration of numerous ice shelves have been observed on the Antarctic Peninsula. The ice shelf in the Prince Gustav Channel retreated gradually since the late 1980's and broke-up in 1995. Tributary glaciers reacted with speed-up, surface lowering and increased ice discharge, consequently contributing to sea level rise. We present a detailed long-term study (1993-2014) on the dynamic response of Sjögren Inlet glaciers to the disintegration of Prince Gustav Ice Shelf. We analyzed various remote sensing datasets to observe the reactions of the glaciers to the loss of the buttressing ice shelf. A strong increase in ice surface velocities was observed with maximum flow speeds reaching 2.82±0.48 m/d in 2007 and 1.50±0.32 m/d in 2004 at Sjögren and Boydell glaciers respectively. Subsequently, the flow velocities decelerated, however in late 2014, we still measured about two times the values of our first measurements in 1996. The tributary glaciers retreated 61.7±3.1 km² behind the former grounding line of the ice shelf. In regions below 1000 m a.s.l., a mean surface lowering of -68±10 m (-3.1 m/a) was observed in the period 1993-2014. The lowering rate decreased to -2.2 m/a in recent years. Based on the surface lowering rates, geodetic mass balances of the glaciers were derived for different time steps. High mass loss rate of -1.21±0.36 Gt/a was found in the earliest period (1993-2001). Due to the dynamic adjustments of the glaciers to the new boundary conditions the ice mass loss reduced to -0.59±0.11 Gt/a in the period 2012-2014, resulting in an average mass loss rate of -0.89±0.16 Gt/a (1993-2014). Including the retreat of the ice front and grounding line, a total mass change of -38.5±7.7 Gt and a contribution to sea level rise of 0.061±0.013 mm were computed. Analysis of the ice flux revealed that available bedrock elevation estimates at Sjögren Inlet are too shallow and are the major uncertainty in ice flux computations. This temporally dense time series analysis of Sjögren Inlet glaciers shows that the adjustments of tributary glaciers to ice shelf disintegration are still going on and provides detailed information of the changes in glacier dynamics.
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[Ser. 1] edited by John Ball; ser.2 by Edward Shirley Kennedy; ser.3, by A.E. Field and Sydney Spencer.
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Bibliography: p. [105]-108.
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"August 1978."
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Bibliographical foot-notes.
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Includes index.