Measurement of the amount and isotopic composition of the CO2 released from the cyanobacterium Synechococcus UTEX 625 after rapid quenching of the active CO2 transport system

1997 ◽  
Vol 75 (6) ◽  
pp. 981-997 ◽  
Author(s):  
Anthony G. Miller ◽  
Christophe Salon ◽  
David T. Canvin ◽  
George S. Espie
Keyword(s):  
Isotopic Composition ◽  
Cell Volume ◽  
Mass Spectrometer ◽  
Transport System ◽  
Time Course ◽  
Rapid Quenching ◽  
Experimental Conditions ◽  
Extracellular Medium ◽  
Fully Depleted ◽  
Entire Cell

Air-grown cells of the cyanobacterium Synechococcus UTEX 625 were suspended in a cuvette connected to a mass spectrometer and supplied with H13C18O3− to investigate the intracellular interconversion between CO2 and HCO3− as determined from the isotopic composition of CO2 appearing in the extracellular medium under a wide variety of experimental conditions. Upon injection of H13C18O3− to the cell suspension in the light, the extracellular [13C16O2] increased. As the CO2 species were 13C labelled, this demonstrated that the 18O-depleted CO2 was originating from the added H13C18O3−. A comparison of the rates of 13C16O16O appearance in the medium with the formation of 13C16O16O from spontaneous dehydration–hydration in the extracellular medium in the presence of cells demonstrated that most of it had to originate from a series of intracellular dehydration–hydration cycles of H13C18O3− that had been recently transported into the cells. During the time course of the experiments both the m/z (mass to charge) = 49 (i.e., 13C18O18O) and 47 (i.e., 13C18O16O) signals decreased constantly, whereas the m/z = 45 signal (i.e.,13C16O2) always increased. Inhibiting CO2 fixation enhanced the amount of CO2 arising in the medium but did not change its isotopic composition, and the CO2 was always fully depleted of 18O. When the CO2 transport system was inhibited by darkening the cells, adding inhibitors such as Na2S or COS, or quenching the uptake of inorganic 13C with an excess of inorganic 12C, the magnitude of the extracellular [13C16O2] was increased but the CO2 species were still always depleted of 18O. Various incubation times of the illuminated cells in the presence of H13C18O3− were used to obtain a variety of internal Ci pool sizes. When the inhibitor (COS) was added, the amount of 13C16O2 arising during the response time of the mass spectrometer was equivalent to the amount of CO2 that would have been present in the whole cell if CO2 and HCO3− were in equilibrium throughout the entire cell volume, but it was at least 40 times higher than the amount of CO2 that would have been present in the cell if the CO2 was confined to the carboxysomes. Experiments were also conducted at pH 9.0 where the spontaneous rate of 13C16O2 production from H13C1803− dehydration–hydration would be negligible, and again the same features were observed. Results show that intracellular HCO3− and CO2 are in rapid equilibrium throughout the entire cell volume. Key words: Synechococcus UTEX 625, cyanobacteria, CO2 leakage, 18O exchange, active CO2 transport, carboxysomes, inorganic C concentrating mechanism.

Simulation of C1 fluxes in cyanobacteria: comparison between the carboxysome hypothesis and the equilibrium proposal

1997 ◽  
Vol 75 (12) ◽  
pp. 2117-2130 ◽  
Author(s):  
Christophe Salon ◽  
David Thomas Canvin
Keyword(s):  
Mathematical Model ◽  
Steady State ◽  
Kinetic Constants ◽  
Experimental Conditions ◽  
Extracellular Medium ◽  
Blue Green Algae ◽  
Permeability Coefficients ◽  
Inorganic Species ◽  
Wide Range ◽  
Entire Cell

Inorganic carbon fluxes were simulated by a mathematical model using an equilibrium hypothesis for a wide range of conditions in a closed system composed of air-grown cells of Synechococcus UTEX 625 in a reaction vessel connected to a mass spectrometer. The metabolic scheme took into account the input fluxes of CO2 and HCO3− transport into the cells, the output fluxes of CO2 and HCO3− efflux, the diversion of Q toward the formation of the internal C2 pool, and photosynthetic CO2 fixation. The equations expressed the variation in concentration of each inorganic species outside and inside the cell as a function of time. The input fluxes were previously characterized by their kinetic constants (K1/2 and Vm) both during initial uptake occurring upon illumination of the cells and under steady-state photosynthesis conditions. The efflux rates of the various Ci species from the cells were investigated under a wide variety of experimental conditions. Using these efflux rates, the permeability coefficients of the cell for CO2 and HCO3− were calculated previously. Using the kinetic constants for CO2 and HCO3− transport, the permeability coefficients of the cell for CO2 and HCO3− and the geometrical characteristics of the cells, the model simulated precisely the [HCO3−]/[CO2] ratio and the [CO2] and [O2] changes in the extracellular medium as well as the rate of filling of the internal Ci pool under various conditions. Accurate fitting of experimental data with calculated values were possible only when the intracellular Ci species were assumed to be in equilibrium throughout the entire cell volume. Results are discussed and compared with those given by previous hypotheses. Key words: Synechococcus UTEX 625, blue green algae, cyanobacteria, mathematical model, active CO2 transport, active HCO3− transport, steady state, photosynthesis, Ci concentrating mechanism.

scholarly journals Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules.

1980 ◽  
Vol 75 (2) ◽  
pp. 109-140 ◽  
Author(s):  
G Salama ◽  
R G Johnson ◽  
A Scarpa
Keyword(s):  
Time Course ◽  
Electrical Potential ◽  
Accurate Method ◽  
Rapid Quenching ◽  
Chromaffin Granules ◽  
Experimental Conditions ◽  
Ph Gradients ◽  
Nernst Potential ◽  
Carbonyl Cyanide ◽  
Granule Membrane

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane-permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3-(5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.

A Simple Method for Estimation of Lipoxygenase and Cyclo-Oxygenase Pathways in Human Platelets - the Use of Thiobarbituric Acid Reaction

1980 ◽  
Vol 44 (02) ◽  
pp. 111-114 ◽  
Author(s):  
Hiroshi Takayama ◽  
Minoru Okuma ◽  
Haruto Uchino
Keyword(s):  
Time Course ◽  
Normal Values ◽  
Human Platelet ◽  
Thiobarbituric Acid ◽  
Human Platelets ◽  
Optimal Conditions ◽  
Experimental Conditions ◽  
Simple Method ◽  
Acid Reaction ◽  
Optimal Ph

SummaryTo develop a simple method for estimation of platelet lipoxygenase (PLO) and cyclo-oxygenase (PCO) pathways, the arachidonic acid (AA) metabolism of human platelet was investigated under various experimental conditions by the use of the thiobarbituric acid (TBA) reaction and a radioisotope technique. A TBA-reactive substance different from malondialdehyde (MDA) via PCO pathway was detected and shown to be derived from the PLO pathway. Since the optimal pH and time course of its formation were different from those of MDA formation via PCO pathway, PLO and PCO pathways were estimated by quantitating the TBA-reactive substances produced by the incubation of AA either with aspirin-treated platelets or with untreated ones, respectively, each under optimal conditions. Normal values expressed in terms of nmol MDA/108 platelets were 1.17±0.34 (M±SD, n = 31) and 0.79±0.15 (n = 31) for PLO and PCO pathways, respectively.

scholarly journals Role of Mammalian Auditory Cortex in the Perception of Elementary Sound Properties

2001 ◽  
Vol 85 (6) ◽  
pp. 2350-2358 ◽  
Author(s):  
Sanjiv K. Talwar ◽  
Pawel G. Musial ◽  
George L. Gerstein
Keyword(s):  
Auditory Cortex ◽  
Time Course ◽  
Auditory Evoked Potentials ◽  
Mammalian Species ◽  
Sound Intensity ◽  
Primary Auditory Cortex ◽  
Normal Hearing ◽  
Auditory Task ◽  
Experimental Conditions

Studies in several mammalian species have demonstrated that bilateral ablations of the auditory cortex have little effect on simple sound intensity and frequency-based behaviors. In the rat, for example, early experiments have shown that auditory ablations result in virtually no effect on the rat's ability to either detect tones or discriminate frequencies. Such lesion experiments, however, typically examine an animal's performance some time after recovery from ablation surgery. As such, they demonstrate that the cortex is not essential for simple auditory behaviors in the long run. Our study further explores the role of cortex in basic auditory perception by examining whether the cortex is normally involved in these behaviors. In these experiments we reversibly inactivated the rat primary auditory cortex (AI) using the GABA agonist muscimol, while the animals performed a simple auditory task. At the same time we monitored the rat's auditory activity by recording auditory evoked potentials (AEP) from the cortical surface. In contrast to lesion studies, the rapid time course of these experimental conditions preclude reorganization of the auditory system that might otherwise compensate for the loss of cortical processing. Soon after bilateral muscimol application to their AI region, our rats exhibited an acute and profound inability to detect tones. After a few hours this state was followed by a gradual recovery of normal hearing, first of tone detection and, much later, of the ability to discriminate frequencies. Surface muscimol application, at the same time, drastically altered the normal rat AEP. Some of the normal AEP components vanished nearly instantaneously to unveil an underlying waveform, whose size was related to the severity of accompanying behavioral deficits. These results strongly suggest that the cortex is directly involved in basic acoustic processing. Along with observations from accompanying multiunit experiments that related the AEP to AI neuronal activity, our results suggest that a critical amount of activity in the auditory cortex is necessary for normal hearing. It is likely that the involvement of the cortex in simple auditory perceptions has hitherto not been clearly understood because of underlying recovery processes that, in the long-term, safeguard fundamental auditory abilities after cortical injury.

Kinetics of reversible DIDS inhibition of chloride self exchange in human erythrocytes

1989 ◽  
Vol 257 (4) ◽  
pp. C601-C606 ◽  
Author(s):  
T. Janas ◽  
P. J. Bjerrum ◽  
J. Brahm ◽  
J. O. Wieth
Keyword(s):  
Transport System ◽  
Time Course ◽  
Order Rate Constant ◽  
Anion Transport ◽  
Disulfonic Acid ◽  
Reversible Inhibitor ◽  
Apparent Dissociation Constant ◽  
Anion Transport System ◽  
Relative Change ◽  
Kinetics Of

The capnophorin (band 3)-mediated chloride self exchange flux in intact erythrocytes and in resealed erythrocyte ghosts was determined at pH 7.3 by measuring the unidirectional efflux of 36Cl-. The time-dependent irreversible inactivation of the anion transport system by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was measured as the relative change of the unidirectional 36Cl efflux rate. The rate of covalent DIDS binding under conditions of excess DIDS in solution that ensure a complete irreversible inhibition followed an exponential time course with a rate coefficient Kcov (min-1). The Arrhenius activation enthalpy of Kcov was constant, 114 kJ/mol, at 0-38 degrees C. At 38 and 0 degrees C, Kcov was 0.5 min-1 [half time (T1/2) = ln2/Kcov = 1.3 min] and 0.004 min-1 (T1/2 = 178 min), respectively. The slow irreversible DIDS binding to the anion transport system at 0 degrees C allows a determination of the kinetics of the reversible DIDS reaction. The pseudo first-order rate constant for binding, kon, was 3.5 X 10(5) (M.s)-1. The apparent dissociation constant, KD, determined from the steady-state binding to the erythrocyte membrane was 3.1 X 10(-8) M at an equal internal and external Cl- concentration of 165 mM (0 degrees C). The value of KD shows that DIDS is the most efficient reversible inhibitor among the stilbene derivatives so far studied. Maximum reversible inhibition by DIDS was obtained by binding of a minimum of approximately 10(6) molecules/cell membrane. The number is similar to that obtained from studies of irreversible DIDS binding.

scholarly journals Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation

2021 ◽  
Author(s):  
Julia L. E. Willett ◽  
Jennifer L. Dale ◽  
Lucy M. Kwiatkowski ◽  
Jennifer L. Powers ◽  
Michelle L. Korir ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Growth Medium ◽  
Biofilm Formation ◽  
Time Course ◽  
Opportunistic Pathogen ◽  
Genetic Determinants ◽  
Experimental Conditions ◽  
Biofilm Reactors ◽  
Biofilm Architecture ◽  
Biofilm Development

AbstractEnterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation.ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

scholarly journals Evaluation of the chemical composition of gas- and particle-phase products of aromatic oxidation

2020 ◽  
Vol 20 (16) ◽  
pp. 9783-9803
Author(s):  
Archit Mehra ◽  
Yuwei Wang ◽  
Jordan E. Krechmer ◽  
Andrew Lambe ◽  
Francesca Majluf ◽  
...  
Keyword(s):  
Gas Phase ◽  
Mass Spectrometer ◽  
Urban Areas ◽  
Minor Component ◽  
Proton Transfer Reaction ◽  
Chemical Mechanism ◽  
Particle Phase ◽  
Experimental Conditions ◽  
Radical Oxidation ◽  
Product Ions

Abstract. Aromatic volatile organic compounds (VOCs) are key anthropogenic pollutants emitted to the atmosphere and are important for both ozone and secondary organic aerosol (SOA) formation in urban areas. Recent studies have indicated that aromatic hydrocarbons may follow previously unknown oxidation chemistry pathways, including autoxidation that can lead to the formation of highly oxidised products. In this study we evaluate the gas- and particle-phase ions measured by online mass spectrometry during the hydroxyl radical oxidation of substituted C9-aromatic isomers (1,3,5-trimethylbenzene, 1,2,4-trimethylbenzene, propylbenzene and isopropylbenzene) and a substituted polyaromatic hydrocarbon (1-methylnaphthalene) under low- and medium-NOx conditions. A time-of-flight chemical ionisation mass spectrometer (ToF-CIMS) with iodide–anion ionisation was used with a filter inlet for gases and aerosols (FIGAERO) for the detection of products in the particle phase, while a Vocus proton-transfer-reaction mass spectrometer (Vocus-PTR-MS) was used for the detection of products in the gas phase. The signal of product ions observed in the mass spectra were compared for the different precursors and experimental conditions. The majority of mass spectral product signal in both the gas and particle phases comes from ions which are common to all precursors, though signal distributions are distinct for different VOCs. Gas- and particle-phase composition are distinct from one another. Ions corresponding to products contained in the near-explicit gas phase Master Chemical Mechanism (MCM version 3.3.1) are utilised as a benchmark of current scientific understanding, and a comparison of these with observations shows that the MCM is missing a range of highly oxidised products from its mechanism. In the particle phase, the bulk of the product signal from all precursors comes from ring scission ions, a large proportion of which are more oxidised than previously reported and have undergone further oxidation to form highly oxygenated organic molecules (HOMs). Under the perturbation of OH oxidation with increased NOx, the contribution of HOM-ion signals to the particle-phase signal remains elevated for more substituted aromatic precursors. Up to 43 % of product signal comes from ring-retaining ions including HOMs; this is most important for the more substituted aromatics. Unique products are a minor component in these systems, and many of the dominant ions have ion formulae concurrent with other systems, highlighting the challenges in utilising marker ions for SOA.

scholarly journals The ionic basis of the hypo-osmotic depolarization in neurons from the opisthobranch mollusc Elysia chlorotica

1992 ◽  
Vol 163 (1) ◽  
pp. 169-186
Author(s):  
R. H. Quinn ◽  
S. K. Pierce
Keyword(s):  
Cell Volume ◽  
Time Course ◽  
The Other ◽  
Resting Potential ◽  
Ion Concentrations ◽  
Elysia Chlorotica ◽  
Apparent Relationship ◽  
Volume Recovery ◽  
Original Potential ◽  
Ionic Basis

The resting potential of identified cells (Parker cells) in the abdominal ganglion of Elysia chlorotica (Gould) depolarizes by about 30 mV in response to a 50% reduction in osmolality and returns to the original potential in 20 min. Cell volume recovery requires approximately 2 h. Thus, recovery of the resting potential is not dependent on recovery of cell volume. The hypo-osmotic depolarization persists following inhibition of the electrogenic Na+/K(+)-ATPase with ouabain, and the levels of extracellular K+ and Cl- have little effect on the magnitude of the depolarization, while decreasing extracellular Na+ concentration produces a depolarization of only 10 mV. This suggests that the hypo-osmotic depolarization in Parker cells results mostly from increased relative permeability to Na+. Following transfer from 920 to 460 mosmol kg-1, Na+, Cl- and proline betaine leave the cells while intracellular K+ is conserved. Loss of intracellular Na+ and conservation of intracellular K+ are dependent on active transport by the Na+/K(+)-ATPase. Na+ and proline betaine leave the cells with a time course that is much longer than that of the hypo-osmotic depolarization. Unlike the other solutes, most of the reduction in intracellular Cl- concentration occurs coincidentally with the hypo-osmotic depolarization. However, unlike the hypo-osmotic depolarization, bulk loss of Cl- does not require the reduction in osmolality, only the reduction in extracellular ion concentrations. There is no apparent relationship between membrane depolarization and the regulation of intracellular osmolytes in Elysia neurons following hypo-osmotic stress.

scholarly journals Formaldehyde and Glyoxal Measurement Deploying a Selected Ion Flow Tube Mass Spectrometer (SIFT-MS)

2021 ◽  
Author(s):  
Antonia Zogka ◽  
Manolis N. Romanias ◽  
Frederic Thevenet
Keyword(s):  
Proton Transfer ◽  
Real Time ◽  
Mass Spectrometer ◽  
Sharp Decrease ◽  
Ionization Mass ◽  
Flow Tube ◽  
Experimental Conditions ◽  
Ion Flow ◽  
Outdoor Environments ◽  
Selected Ion Flow Tube

Abstract. Formaldehyde (FM) and glyoxal (GL) are important atmospheric species of indoor and outdoor environments. They are either directly emitted in the atmosphere or they are formed through the oxidation of organic compounds by indoor and/or outdoor atmospheric oxidants. Despite their importance, the real-time monitoring of these compounds with soft ionization mass spectrometric techniques, e.g. proton transfer mass spectrometry (PTR-MS), remains problematic and is accompanied by low sensitivity. In this study, we evaluate the performance of a multi-ion selected ion flow tube mass spectrometer (SIFT-MS) to monitor in real-time atmospherically relevant concentrations of FM and GL under controlled experimental conditions. The SIFT-MS used is operated under standard conditions (SC), as proposed by the supplier, and customized conditions (CC), to achieve higher sensitivity. In the case of FM, SIFT-MS sensitivity is marginally impacted by RH, and the detection limits achieved are below 200 ppt. Contrariwise, in the case of GL, a sharp decrease of instrument sensitivity is observed with increasing RH when the H3O+ ion is used. Nevertheless, the detection of GL using NO+ precursor ion is moderately impacted by moisture with an actual positive sensitivity response. Therefore, we recommend the use of NO+ precursor for reliable detection and quantitation of GL. This work evidences that SIFT-MS can be considered as an efficient tool to monitor the concentration of FM and GL using SIFT-MS in laboratory experiments and potentially in indoor or outdoor environments. Furthermore, SIFT-MS technology still allows great possibilities for sensitivity improvement and high potential for monitoring low proton transfer affinity compounds.

Anaerobiosis: A Possible Source of Osmotic Solute for High-salinity Acclimation in Marine Molluscs

1975 ◽  
Vol 62 (3) ◽  
pp. 589-598
Author(s):  
RICHARD M. BAGINSKI ◽  
SIDNEY K. PIERCE
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Volume ◽  
Free Amino ◽  
Time Course ◽  
High Salinity ◽  
Gulf Coast ◽  
Total Amino Acid ◽  
Salinity Acclimation ◽  
Rapid Accumulation

1. When stressed with high-salinity exposure, cell volume is restored in ventricles of Modiolus demissus demissus by a rapid accumulation of intracellular free amino acids. 2. Although the total amino acid pool increases and remains at a constant high level thereafter, the pattern and time course of accumulation is different for each major amino acid (glycine, alanine, taurine, and proline). 3. Initially, cell volume is restored by a rapid accumulation of alanine, but later its concentration decreases while glycine and taurine accumulate. Although at first not detected, the proline concentration increases, peaks and subsequently disappears again. 4. Isolated ventricles recover normal activity after large environmental salinity increases. 5. During recovery the intracellular free amino acid changes in isolated ventricles are similar to the initial pattern of accumulation in whole animals, i.e., alanine, and to a lesser extent, proline and glycine accumulate. 6. Finally, isolated ventricles undergo a period of decreased oxygen consumption on exposure to an increased salinity. 7. These results suggest that the initial stages of high-salinity acclimation in molluscs depends upon the synthesis of amino acids via a known anaerobic biochemical pathway. Note: Contribution No. 33 from the Tallahassee, Sopchoppy and Gulf Coast Marine Biological Association.

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