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Search Results: 1 - 10 of 543436 matches for " Ryan A. V. Bell "
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Insights into the In Vivo Regulation of Glutamate Dehydrogenase from the Foot Muscle of an Estivating Land Snail
Ryan A. V. Bell,Neal J. Dawson,Kenneth B. Storey
Enzyme Research , 2012, DOI: 10.1155/2012/317314
Abstract: Land snails, Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation. 1. Introduction Glutamate dehydrogenase (GDH; E.C. 1.4.1.3) is an important enzyme that contributes to a diverse set of metabolic processes. GDH catalyzes the following reversible reaction within the mitochondrial matrix: Through oxidative deamination, GDH gates the entry of numerous amino acid carbon skeletons into the Krebs cycle for increased energy production or gluconeogenic output. Furthermore, GDH-derived ammonium ions provide the primary source of nitrogen for the synthesis of urea via the urea cycle. In the reverse direction, GDH acts to synthesize L-glutamate for use in protein synthesis or, alternatively, transamination reactions. Given the importance of GDH in both carbohydrate and nitrogen metabolism, it was hypothesized to be a critical enzyme in animals that experience drastic alterations to cellular biochemistry in response to harsh environmental conditions. Animals that live in seasonally hot and dry environments usually require some mechanism to survive periodic droughts and the scarcity of food that typically follows. One such mechanism is estivation, which is a state of aerobic torpor that is employed by a range of organisms including amphibians, reptiles, small mammals, and land snails [1]. Estivation entails major behavioral, physiological, and biochemical adaptations that allow for prolonged survival under harsh conditions. Particularly important for this
Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans
Neal J. Dawson,Ryan A. V. Bell,Kenneth B. Storey
Enzyme Research , 2013, DOI: 10.1155/2013/784973
Abstract:
Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans
Neal J. Dawson,Ryan A. V. Bell,Kenneth B. Storey
Enzyme Research , 2013, DOI: 10.1155/2013/784973
Abstract: Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20?h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) for L-lactate and a higher value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions. 1. Introduction Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a critical enzyme involved in anaerobic metabolism. LDH catalyzes the following reversible reaction: In this capacity, LDH favors the pyruvate reducing direction in skeletal muscle tissue, converting the glycolytic end product to lactate and regenerating the NAD+ pools to maintain a prolonged glycolytic flux [1]. This process is especially critical to those organisms that enter periodically into hypoxic/anoxic environments, where maintaining NAD+/NADH balance is essential for ATP production. Under low oxygen insult, organisms often rely solely on the glycolytic pathway to produce ATP. The greatly reduced production of ATP via glycolysis, as compared to that of oxidative phosphorylation, results in difficult challenges for anoxia-tolerant organisms to overcome. Several of these organisms employ alternate anaerobic pathways to increase ATP yield and/or depress their metabolic rate to survive the low oxygen stress [2]. Furthermore, these organisms typically need to safeguard against the accumulation of acidic glycolytic end products such as lactate, which disrupts cellular homeostasis throughout prolonged exposure to anoxia [2]. Freshwater turtles, Trachemys scripta elegans, have demonstrated a remarkable ability to survive submerged in cold water for 4-5 months during the winter to escape freezing air temperatures. While submerged,
Glucose-6-phosphate dehydrogenase regulation in the hepatopancreas of the anoxia-tolerant marine mollusc, Littorina littorea
Judeh L. Lama,Ryan A.V. Bell,Kenneth B. Storey
PeerJ , 2013, DOI: 10.7717/peerj.21
Abstract: Glucose-6-phosphate dehydrogenase (G6PDH) gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions. Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8) showed a 38% decrease in Km G6P and enhanced inhibition by urea, whereas in pH 6 assays Km NADP and maximal activity changed significantly between the two states. The mechanism underlying anoxia-responsive changes in enzyme properties proved to be a change in the phosphorylation state of G6PDH. This was documented with immunoblotting using an anti-phosphoserine antibody, in vitro incubations that stimulated endogenous protein kinases versus protein phosphatases and significantly changed Km G6P, and phosphorylation of the enzyme with 32P-ATP. All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG) and protein phosphatases (PP1 or PP2C). Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the transition back to aerobic conditions when the reintroduction of oxygen causes a rapid increase in oxidative stress.
Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism
Ali Shahriari,Neal J. Dawson,Ryan A. V. Bell,Kenneth B. Storey
Enzyme Research , 2013, DOI: 10.1155/2013/461374
Abstract: The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24?h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves. 1. Introduction Lactate dehydrogenase catalyzes the reversible conversion of pyruvate to lactate, with the concomitant oxidation of NADH to NAD+. Under anaerobic conditions, LDH becomes an important enzyme due to its ability to regenerate NAD+ and allows for continued carbon flow through the glycolytic pathway to support anaerobic ATP synthesis [1]. This process can be especially important in those organisms that are exposed to hypoxic or anoxic conditions for extended periods of time and require energy balance to be maintained solely through the functioning of glycolysis. Littorina littorea are marine molluscs that are native to the intertidal zones of the Atlantic coast of Europe (from Scandinavia to Spain) and have been introduced to the east coast of North America as well as several other locations around the world. Changing tides frequently expose these gill-breathing snails to prolonged oxygen deprivation at low tide [2]. Moreover, environmental conditions, such as high salinity, predation, or water pollutants can cause the snails to shut their shell openings, which over an extended period of time can also generate an anoxic exposure [3, 4]. In order to survive
Purification and properties of glyceraldehyde-3-phosphate dehydrogenase from the skeletal muscle of the hibernating ground squirrel, Ictidomys tridecemlineatus
Ryan A.V. Bell,Jeffrey C. Smith,Kenneth B. Storey
PeerJ , 2015, DOI: 10.7717/peerj.634
Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the skeletal muscle of euthermic and torpid Ictidomys tridecemlineatus was purified to electrophoretic homogeneity using a novel method involving Blue-agarose and Phenyl-agarose chromatography. Kinetic analysis of the enzymes isolated from the two conditions suggested the existence of two structurally distinct proteins, with GAPDH V max being 40–60% less for the enzyme from the torpid condition (in both glycolytic and gluconeogenic directions) as compared to the euthermic enzyme form. Thermal denaturation, in part determined by differential scanning fluorimetry, revealed that purified GAPDH from the torpid animals was significantly more stable that the enzyme from the euthermic condition. Mass spectrometry combined with Western blot analyses of purified GAPDH indicate that the cellular GAPDH population is extensively modified, with posttranslational phosphorylation, acetylation and methylation being detected. Global reduction in GAPDH tyrosine phosphorylation during torpor as well as site specific alterations in methylation sites suggests that that the stable changes observed in kinetic and structural GAPDH properties may be due to posttranslational modification of this enzyme during torpor. Taken together, these results suggest a stable suppression of GAPDH (possibly by some reversible posttranslational modification) during ground squirrel torpor, which likely contributes to the overall reduction in carbohydrate metabolism when these animals switch to lipid fuels during dormancy.
Short period forecasting of catchment-scale precipitation. Part II: a water-balance storm model for short-term rainfall and flood forecasting
V. A. Bell,R. J. Moore
Hydrology and Earth System Sciences (HESS) & Discussions (HESSD) , 2000,
Abstract: A simple two-dimensional rainfall model, based on advection and conservation of mass in a vertical cloud column, is investigated for use in short-term rainfall and flood forecasting at the catchment scale under UK conditions. The model is capable of assimilating weather radar, satellite infra-red and surface weather observations, together with forecasts from a mesoscale numerical weather prediction model, to obtain frequently updated forecasts of rainfall fields. Such data assimilation helps compensate for the simplified model dynamics and, taken together, provides a practical real-time forecasting scheme for catchment scale applications. Various ways are explored for using information from a numerical weather prediction model (16.8 km grid) within the higher resolution model (5 km grid). A number of model variants is considered, ranging from simple persistence and advection methods used as a baseline, to different forms of the dynamic rainfall model. Model performance is assessed using data from the Wardon Hill radar in Dorset for two convective events, on 10 June 1993 and 16 July 1995, when thunderstorms occurred over southern Britain. The results show that (i) a simple advection-type forecast may be improved upon by using multiscan radar data in place of data from the lowest scan, and (ii) advected, steady-state predictions from the dynamic model, using 'inferred updraughts', provides the best performance overall. Updraught velocity is inferred at the forecast origin from the last two radar fields, using the mass-balance equation and associated data and is held constant over the forecast period. This inference model proves superior to the buoyancy parameterisation of updraught employed in the original formulation. A selection of the different rainfall forecasts is used as input to a catchment flow forecasting model, the IH PDM (Probability Distributed Moisture) model, to assess their effect on flow forecast accuracy for the 135 km2 Brue catchment in Somerset. Keywords: rainfall forecasting, flood forecasting, weather radar, satellite, storm model
The sensitivity of catchment runoff models to rainfall data at different spatial scales
V. A. Bell,R. J. Moore
Hydrology and Earth System Sciences (HESS) & Discussions (HESSD) , 2000,
Abstract: The sensitivity of catchment runoff models to rainfall is investigated at a variety of spatial scales using data from a dense raingauge network and weather radar. These data form part of the HYREX (HYdrological Radar EXperiment) dataset. They encompass records from 49 raingauges over the 135 km2 Brue catchment in south-west England together with 2 and 5 km grid-square radar data. Separate rainfall time-series for the radar and raingauge data are constructed on 2, 5 and 10 km grids, and as catchment average values, at a 15 minute time-step. The sensitivity of the catchment runoff models to these grid scales of input data is evaluated on selected convective and stratiform rainfall events. Each rainfall time-series is used to produce an ensemble of modelled hydrographs in order to investigate this sensitivity. The distributed model is shown to be sensitive to the locations of the raingauges within the catchment and hence to the spatial variability of rainfall over the catchment. Runoff sensitivity is strongest during convective rainfall when a broader spread of modelled hydrographs results, with twice the variability of that arising from stratiform rain. Sensitivity to rainfall data and model resolution is explored and, surprisingly, best performance is obtained using a lower resolution of rainfall data and model. Results from the distributed catchment model, the Simple Grid Model, are compared with those obtained from a lumped model, the PDM. Performance from the distributed model is found to be only marginally better during stratiform rain (R2 of 0.922 compared to 0.911) but significantly better during convective rain (R2 of 0.953 compared to 0.909). The improved performance from the distributed model can, in part, be accredited to the excellence of the dense raingauge network which would not be the norm for operational flood warning systems. In the final part of the paper, the effect of rainfall resolution on the performance of the 2 km distributed model is explored. The need to recalibrate the model for use with rainfall data of a given resolution, particularly for periods of convective rain, is highlighted. Again, best performance is obtained using lower resolution rainfall data. This is interpreted as evidence for the need to improve the distributed model structure to make better use of the higher resolution information on rainfall and topographic controls on runoff. Degrading the resolution of rainfall data, model or both to achieve the smoothing apparently needed is not seen as wholly appropriate. Keywords: rainfall, runoff, sensitivity, scale, mod
Incorporation of groundwater losses and well level data in rainfall-runoff models illustrated using the PDM
R. J. Moore,V. A. Bell
Hydrology and Earth System Sciences (HESS) & Discussions (HESSD) , 2002,
Abstract: Intermittent streamflow is a common occurrence in permeable catchments, especially where there are pumped abstractions to water supply. Many rainfall-runoff models are not formulated so as to represent ephemeral streamflow behaviour or to allow for the possibility of negative recharge arising from groundwater pumping. A groundwater model component is formulated here for use in extending existing rainfall-runoff models to accommodate such ephemeral behaviour. Solutions to the Horton-Izzard equation resulting from the conceptual model of groundwater storage are adapted and the form of nonlinear storage extended to accommodate negative inputs, water storage below which outflow ceases, and losses to external springs and underflows below the gauged catchment outlet. The groundwater model component is demonstrated through using it as an extension of the PDM rainfall-runoff model. It is applied to the River Lavant, a catchment in Southern England on the English Chalk, where it successfully simulates the ephemeral streamflow behaviour and flood response together with well level variations. Keywords: groundwater, rainfall-runoff model, ephemeral stream, well level, spring, abstraction
A grid-based distributed flood forecasting model for use with weather radar data: Part 1. Formulation
V. A. Bell,R. J. Moore
Hydrology and Earth System Sciences (HESS) & Discussions (HESSD) , 1998,
Abstract: A practical methodology for distributed rainfall-runoff modelling using grid square weather radar data is developed for use in real-time flood forecasting. The model, called the Grid Model, is configured so as to share the same grid as used by the weather radar, thereby exploiting the distributed rainfall estimates to the full. Each grid square in the catchment is conceptualised as a storage which receives water as precipitation and generates water by overflow and drainage. This water is routed across the catchment using isochrone pathways. These are derived from a digital terrain model assuming two fixed velocities of travel for land and river pathways which are regarded as model parameters to be optimised. Translation of water between isochrones is achieved using a discrete kinematic routing procedure, parameterised through a single dimensionless wave speed parameter, which advects the water and incorporates diffusion effects through the discrete space-time formulation. The basic model routes overflow and drainage separately through a parallel system of kinematic routing reaches, characterised by different wave speeds but using the same isochrone-based space discretisation; these represent fast and slow pathways to the basin outlet, respectively. A variant allows the slow pathway to have separate isochrones calculated using Darcy velocities controlled by the hydraulic gradient as estimated by the local gradient of the terrain. Runoff production within a grid square is controlled by its absorption capacity which is parameterised through a simple linkage function to the mean gradient in the square, as calculated from digital terrain data. This allows absorption capacity to be specified differently for every grid square in the catchment through the use of only two regional parameters and a DTM measurement of mean gradient for each square. An extension of this basic idea to consider the distribution of gradient within the square leads analytically to a Pareto distribution of absorption capacity, given a power distribution of gradient within the square. The probability-distributed model theory (Moore, 1985) can then be used directly to obtain the integrated runoff production for the square for routing to the catchment outlet. justification for the simple linkage function is in part sought through consideration of variants on the basic model where (i) runoff production is based on a topographic index control on saturation and (ii) absorption capacity is related to the Integrated Air Capacity of the soil, as obtained from soil survey. An impervious area frac
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