scholarly journals A pyruvate–proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle

2000 ◽  
Vol 346 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Nicole VANDERHEYDEN ◽  
Julian WONG ◽  
Roberto DOCAMPO
Keyword(s):  
Steady State ◽  
Trypanosoma Brucei ◽  
Intracellular Ph ◽  
Fluorescent Dyes ◽  
Minor Role ◽  
Proton Efflux ◽  
Acetoxymethyl Ester ◽  
Wide Range ◽  
Atpase Inhibitors ◽  
Recovery From Acidification

Regulation of intracellular pH (pHi) and H+ efflux were investigated in Trypanosoma brucei bloodstream and procyclic trypomastigotes using the fluorescent dyes 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester and free BCECF respectively. pHi in bloodstream and procyclic trypomastigotes was 7.47±0.06 and 7.53±0.07 respectively. Differences in the mechanisms for the regulation of pHi were noted between bloodstream and procyclic forms. Procyclic trypomastigotes maintained their pHi at neutral over a wide range of external pH values from 6 to 8, and in the absence of K+ or Na+. The H+-ATPase inhibitors N,Nʹ-dicyclohexylcarbodi-imide (DCCD), diethylstilboestrol and N-ethylmaleimide substantially decreased the steady-state pHi and inhibited its recovery from acidification. The rate of H+ efflux in these forms was determined to be 62±6.5 nmol/min per mg of protein, and was substantially decreased by H+-ATPase inhibitors. The data support the presence of an H+-ATPase as the major regulator of pHi in procyclic trypomastigotes. In contrast, bloodstream trypomastigotes were unable to maintain a neutral pH under acidic conditions, and their steady-state pHi and recovery from acidification were unaffected by H+-ATPase inhibitors, except for DCCD (100 μM). Their steady-state pHi was markedly decreased in glucose-free buffer or by ≥ 10 mM pyruvate, whereas procyclic trypomastigotes were unaffected by similar treatments. The rate of H+ efflux in bloodstream trypomastigotes was 534±38 nmol/min per mg of protein, and was decreased in the absence of glucose and by the addition of pyruvate or DCCD. Pyruvate efflux in these forms was calculated to be 499±34 nmol/min per mg of protein, and was significantly inhibited by DCCD, 4,4ʹ-di-isothiocyanatodihydrostilbene-2,2ʹ-disulphonic acid and α-cyanohydroxycinnamic acid. The pyruvate analogues β-hydroxypyruvate, 3-bromopyruvate, 3-oxoglutarate, oxaloacetate, 3-oxoisovalerate and 3-oxoisohexanoate significantly decreased pHi, as well as proton and pyruvate efflux, whereas lactate had only a small effect, and no effect was observed with citrate or fumarate. The inhibition by pyruvate analogues of pyruvate efflux, proton efflux and acidification of pHi supports the hypothesis that pyruvate efflux is accompanied by proton efflux and that this is the major pHi control mechanism in bloodstream forms. Inhibition by H+-ATPase inhibitors of residual H+ efflux in the absence of glucose or in the presence of high extracellular pyruvate indicates a minor role for H+-ATPase(s) in control of pHi in bloodstream forms.

scholarly journals Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange.

1990 ◽  
Vol 96 (6) ◽  
pp. 1247-1269 ◽  
Author(s):  
S Liu ◽  
D Piwnica-Worms ◽  
M Lieberman
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Ph Regulation ◽  
Heart Cells ◽  
Efflux Rate Constant ◽  
Recovery From Acidification ◽  
Acid Load ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
Phi Regulation

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.

scholarly journals Adrenalin-Induced Na+/H+ Exchange in' Trout Erythrocytes and its Effects Upon Oxygen-Carrying Capacity

1985 ◽  
Vol 118 (1) ◽  
pp. 229-246 ◽  
Author(s):  
A. R. COSSINS ◽  
P. A. RICHARDSON
Keyword(s):  
Steady State ◽  
Intracellular Ph ◽  
Carrying Capacity ◽  
Extracellular Ph ◽  
Extracellular Medium ◽  
Root Effect ◽  
Fish Blood ◽  
Wide Range ◽  
Oxygen Carrying Capacity ◽  
Normal Relationship

Addition of adrenalin (10−4moll−1) to trout erythrocytes in an unbuffered saline resulted in a rapid acidification of the extracellular medium. This process was inhibited by amiloride (K½10−4moll−1) and by the removal of extracellular Na+. The rate of acidification was a saturable function of extracellular Na+ concentration. When extracellular pH was maintained constant by continual titration with KOH, adrenalin induced a transient burst of H+ efflux. During this period the loss of cellular H+ equivalents was approximately equal to the net gain of Na+, providing evidence for a Na+/H+ exchange with a stoichiometry of 1. The steady state following stimulation with adrenalin could be disturbed by changes in extracellular pH. After the addition of adrenalin, intracellular pH (pHi) was increased by 0.2-0.3 units but did not exceed extracellular pH, as required if the Na+ and H+ concentration ratios came into equilibrium. The increase in pHi in stimulated compared with control cells was maintained approximately constant over a wide range of pHo, suggesting that pH equilibration by the Jacob-Stewart cycle was operating normally and that the activation of Na+/H+ exchange provides an offset to the normal relationship between pHi and pHo. The steady state results from a balance of an increase Na+/H+ and CI−/HCO3− exchange with an increased rate of Na+ pumping and next KCl efflux. In a buffered saline, adrenalin caused a 22–46% increase in the oxygen-carrying capacity of trout erythrocytes. It is suggested that this was due to a Root effect of trout haemoglobin caused by cellular alkalinization when the Na+/H+ exchange mechanism was activiated. This observation suggests that many published values for oxygencarrying capacity of fish blood require re-evaluation.

Spectroscopic Studies of Asparaginyl-tRNA Synthetase from Entamoeba histolytica

2019 ◽  
Vol 26 (6) ◽  
pp. 435-448
Author(s):  
Priyanka Biswas ◽  
Dillip K. Sahu ◽  
Kalyanasis Sahu ◽  
Rajat Banerjee
Keyword(s):  
Circular Dichroism ◽  
Steady State ◽  
Entamoeba Histolytica ◽  
Protein Concentration ◽  
Trna Synthetase ◽  
Solvent Exposure ◽  
Steady State Fluorescence ◽  
State Process ◽  
Wide Range ◽  
Circular Dichroïsm

Background: Aminoacyl-tRNA synthetases play an important role in catalyzing the first step in protein synthesis by attaching the appropriate amino acid to its cognate tRNA which then transported to the growing polypeptide chain. Asparaginyl-tRNA Synthetase (AsnRS) from Brugia malayi, Leishmania major, Thermus thermophilus, Trypanosoma brucei have been shown to play an important role in survival and pathogenesis. Entamoeba histolytica (Ehis) is an anaerobic eukaryotic pathogen that infects the large intestines of humans. It is a major cause of dysentery and has the potential to cause life-threatening abscesses in the liver and other organs making it the second leading cause of parasitic death after malaria. Ehis-AsnRS has not been studied in detail, except the crystal structure determined at 3 Å resolution showing that it is primarily α-helical and dimeric. It is a homodimer, with each 52 kDa monomer consisting of 451 amino acids. It has a relatively short N-terminal as compared to its human and yeast counterparts. Objective: Our study focusses to understand certain structural characteristics of Ehis-AsnRS using biophysical tools to decipher the thermodynamics of unfolding and its binding properties. Methods: Ehis-AsnRS was cloned and expressed in E. coli BL21DE3 cells. Protein purification was performed using Ni-NTA affinity chromatography, following which the protein was used for biophysical studies. Various techniques such as steady-state fluorescence, quenching, circular dichroism, differential scanning fluorimetry, isothermal calorimetry and fluorescence lifetime studies were employed for the conformational characterization of Ehis-AsnRS. Protein concentration for far-UV and near-UV circular dichroism experiments was 8 µM and 20 µM respectively, while 4 µM protein was used for the rest of the experiments. Results: The present study revealed that Ehis-AsnRS undergoes unfolding when subjected to increasing concentration of GdnHCl and the process is reversible. With increasing temperature, it retains its structural compactness up to 45ºC before it unfolds. Steady-state fluorescence, circular dichroism and hydrophobic dye binding experiments cumulatively suggest that Ehis-AsnRS undergoes a two-state transition during unfolding. Shifting of the transition mid-point with increasing protein concentration further illustrate that dissociation and unfolding processes are coupled indicating the absence of any detectable folded monomer. Conclusion: This article indicates that GdnHCl induced denaturation of Ehis-AsnRS is a two – state process and does not involve any intermediate; unfolding occurs directly from native dimer to unfolded monomer. The solvent exposure of the tryptophan residues is biphasic, indicating selective quenching. Ehis-AsnRS also exhibits a structural as well as functional stability over a wide range of pH.

scholarly journals Identification of DC Thermal Steady-State Differential Inductance of Ferrite Power Inductors

Energies ◽  
2021 ◽  
Vol 14 (13) ◽  
pp. 3854
Author(s):  
Salvatore Musumeci ◽  
Luigi Solimene ◽  
Carlo Stefano Ragusa
Keyword(s):  
Steady State ◽  
Input Voltage ◽  
Switching Frequency ◽  
Ohmic Losses ◽  
Steady State Temperature ◽  
Wide Range ◽  
Average Current ◽  
Power Inductors ◽  
Saturable Inductor ◽  
Thermal Steady State

In this paper, we propose a method for the identification of the differential inductance of saturable ferrite inductors adopted in DC–DC converters, considering the influence of the operating temperature. The inductor temperature rise is caused mainly by its losses, neglecting the heating contribution by the other components forming the converter layout. When the ohmic losses caused by the average current represent the principal portion of the inductor power losses, the steady-state temperature of the component can be related to the average current value. Under this assumption, usual for saturable inductors in DC–DC converters, the presented experimental setup and characterization method allow identifying a DC thermal steady-state differential inductance profile of a ferrite inductor. The curve is obtained from experimental measurements of the inductor voltage and current waveforms, at different average current values, that lead the component to operate from the linear region of the magnetization curve up to the saturation. The obtained inductance profile can be adopted to simulate the current waveform of a saturable inductor in a DC–DC converter, providing accurate results under a wide range of switching frequency, input voltage, duty cycle, and output current values.

scholarly journals Humic acids trigger the weak acids stress response in maize seedlings

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Daiane Carvalho Baía ◽  
Fábio L. Olivares ◽  
Daniel B. Zandonadi ◽  
Cleiton de Paula Soares ◽  
Riccardo Spaccini ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Stress Response ◽  
Intracellular Ph ◽  
Humic Acids ◽  
Abiotic Stress Tolerance ◽  
Molecular Response ◽  
Maize Seedlings ◽  
Weak Acids ◽  
Acetoxymethyl Ester ◽  
Chemical Priming

Abstract Background Plants primed by humic acids showed physiological and molecular response against different abiotic stresses without the presence of stressor agents (salinity, drought, heavy metal toxicity). It is plausible that humic acids themselves can act as chemical priming substances in plants. We hypothesized that humic acids can trigger the weak acids stress response in cell plants acidifying the cytosol and thus eliciting the transduction signalling response cascade. Methods The dose–response curves of maize seedlings roots with different concentrations of humic, acetic and salicylic acids determined the most active and inhibitory concentration. These data were further used to evaluate changes on intracellular pH using BCECF-AM probe (2,7-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein, acetoxymethyl ester) and differential transcription level of genes related to weak stress response in plants by qPCR real time. Results Humic acids like short chain organic acids decrease the intracellular pH showed by the increased fluorescence of BCECF probe. The drop in cytosolic pH promoted by humic acids was not transient. We observed a high level of protein kinases related to cell energy-sensing and transcription factors associated to transduction of stress signalling. Conclusion The humic acids can be considered as a chemical priming agent, since in the appropriate concentration they can induce the typical plant abiotic stress response of weak acids inducing plant acclimation and enhancing the abiotic stress tolerance.

SET-VALUED PERFORMANCE APPROXIMATIONS FOR THE QUEUE GIVEN PARTIAL INFORMATION

2020 ◽  
pp. 1-23
Author(s):  
Yan Chen ◽  
Ward Whitt
Keyword(s):  
Steady State ◽  
Decay Rate ◽  
Waiting Time ◽  
Partial Information ◽  
Upper And Lower Bounds ◽  
Interarrival Time ◽  
Limited Information ◽  
Wide Range ◽  
The Mean ◽  
Third Moment

In order to understand queueing performance given only partial information about the model, we propose determining intervals of likely values of performance measures given that limited information. We illustrate this approach for the mean steady-state waiting time in the $GI/GI/K$ queue. We start by specifying the first two moments of the interarrival-time and service-time distributions, and then consider additional information about these underlying distributions, in particular, a third moment and a Laplace transform value. As a theoretical basis, we apply extremal models yielding tight upper and lower bounds on the asymptotic decay rate of the steady-state waiting-time tail probability. We illustrate by constructing the theoretically justified intervals of values for the decay rate and the associated heuristically determined interval of values for the mean waiting times. Without extra information, the extremal models involve two-point distributions, which yield a wide range for the mean. Adding constraints on the third moment and a transform value produces three-point extremal distributions, which significantly reduce the range, producing practical levels of accuracy.

scholarly journals Modelling the atmosphere of lava planet K2-141b: implications for low- and high-resolution spectroscopy

2020 ◽  
Vol 499 (4) ◽  
pp. 4605-4612
Author(s):  
T Giang Nguyen ◽  
Nicolas B Cowan ◽  
Agnibha Banerjee ◽  
John E Moores
Keyword(s):  
Steady State ◽  
Ocean Circulation ◽  
Scale Height ◽  
High Resolution Spectroscopy ◽  
Return Flow ◽  
High Dispersion ◽  
Magma Ocean ◽  
Steady State Flow ◽  
Wide Range ◽  
Very High

ABSTRACT Transit searches have uncovered Earth-size planets orbiting so close to their host star that their surface should be molten, so-called lava planets. We present idealized simulations of the atmosphere of lava planet K2-141b and calculate the return flow of material via circulation in the magma ocean. We then compare how pure Na, SiO, or SiO2 atmospheres would impact future observations. The more volatile Na atmosphere is thickest followed by SiO and SiO2, as expected. Despite its low vapour pressure, we find that a SiO2 atmosphere is easier to observe via transit spectroscopy due to its greater scale height near the day–night terminator and the planetary radial velocity and acceleration are very high, facilitating high dispersion spectroscopy. The special geometry that arises from very small orbits allows for a wide range of limb observations for K2-141b. After determining the magma ocean depth, we infer that the ocean circulation required for SiO steady-state flow is only 10−4 m s−1, while the equivalent return flow for Na is several orders of magnitude greater. This suggests that a steady-state Na atmosphere cannot be sustained and that the surface will evolve over time.

Control of Steady-state Intracellular pH in Intact Perfused Ferret Hearts

1994 ◽  
Vol 26 (7) ◽  
pp. 821-829 ◽  
Author(s):  
Hideo Kusuoka ◽  
Eduardo Marban ◽  
Horacio E. Cingolani
Keyword(s):  
Steady State ◽  
Intracellular Ph

scholarly journals Foslevodopa/Foscarbidopa Is Well Tolerated and Maintains Stable Levodopa and Carbidopa Exposure Following Subcutaneous Infusion

2021 ◽  
pp. 1-8
Author(s):  
Matthew Rosebraugh ◽  
Wei Liu ◽  
Melina Neenan ◽  
Maurizio F. Facheris
Keyword(s):  
Steady State ◽  
Phase 1 ◽  
The United States ◽  
Therapeutic Window ◽  
Fixed Dose ◽  
Advanced Parkinson’S Disease ◽  
Subcutaneous Infusion ◽  
Study Results ◽  
Wide Range ◽  
Favorable Safety

Background: Foslevodopa/foscarbidopa, formerly known as ABBV-951, is a formulation of levodopa/carbidopa prodrugs with solubility that allows for subcutaneous (SC) infusion and is in development for the treatment of motor complications for patients with advanced Parkinson’s disease (aPD). Objective: The current work characterizes the levodopa (LD) and carbidopa (CD) pharmacokinetics (PK) following SC infusions of foslevodopa/foscarbidopa delivered at four different infusion rates in PD patients. Methods: This was a Phase 1, single ascending dose, single-blind study conducted in 28 adult male and female subjects at seven sites in the United States. Foslevodopa/foscarbidopa was administered via abdominal SC infusion in PD patients over 72 hours. Patients were stratified in 4 groups and received a fixed dose of foslevodopa/foscarbidopa based on their oral daily LD intake. Serial plasma PK samples were collected to assay for LD and CD concentrations. Safety and tolerability were assessed throughout the study. Results: LD exposure quickly reached steady state and remained stable with minimal fluctuations. Foslevodopa/foscarbidopa infusion provides stable LD and CD exposures compared to oral LD/CD dosing with the average steady-state exposure ranging from 747-4660 ng/mL for the different groups. Conclusion: Foslevodopa/foscarbidopa was able to provide stable LD and CD exposures in PD patients over 72 hours via SC route of delivery with very low fluctuation in LD concentration level across a wide range of clinically relevant exposures. Foslevodopa/foscarbidopa had a favorable safety profile. The low PK fluctuation following foslevodopa/foscarbidopa infusion is expected to maintain LD exposure to treat aPD patients within a narrow therapeutic window.

Study of Temperature Effect on Servovalve-Controlled Fuel Metering Unit

2015 ◽  
Vol 137 (6) ◽  
Author(s):  
Bin Wang ◽  
Haocen Zhao ◽  
Ling Yu ◽  
Zhifeng Ye
Keyword(s):  
Steady State ◽  
Temperature Variation ◽  
Dynamic Characteristics ◽  
Numerical Study ◽  
Step Response ◽  
Fuel Temperature ◽  
Wide Range ◽  
Fuel Rate ◽  
Testing Condition ◽  
The Given

It is usual that fuel system of an aero-engine operates within a wide range of temperatures. As a result, this can have effect on both the characteristics and precision of fuel metering unit (FMU), even on the performance and safety of the whole engine. This paper provides theoretical analysis of the effect that fluctuation of fuel temperature has on the controllability of FMU and clarifies the drawbacks of the pure mathematical models considering fuel temperature variation for FMU. Taking the electrohydraulic servovalve-controlled FMU as the numerical study, simulation in AMESim is carried out by thermal hydraulic model under the temperatures ranged from −10 to 60 °C to confirm the effectiveness and precision of the model on the basis of steady-state and dynamic characteristics of FMU. Meanwhile, the FMU testing workbench with temperature adjustment device employing the fuel cooler and heater is established to conduct an experiment of the fuel temperature characteristics. Results show that the experiment matches well with the simulation with a relative error no more than 5% and that 0–50 °C fuel temperature variation produces up to 5.2% decrease in fuel rate. In addition, step response increases with the fuel temperature. Fuel temperature has no virtual impact on the steady-state and dynamic characteristics of FMU under the testing condition in this paper, implying that FMU can operate normally in the given temperature range.

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