pH Perturbation Research Articles

Accumulated Oxygen Deficit & pH Recovery during Multiple Bouts of Intense Exercise

PURPOSE: The purpose of this study was to profile the effect of active vs. passive recovery on accumulated oxygen deficit (AOD) and recovery pH during multiple bouts of intense exercise. METHODS: Ten highly trained male cyclists completed two exercise trials. Each trial consisted of 3 exercise bouts to exhaustion (110% workload max) with either a 12 min active (20% workload max) or passive recovery between bouts. AOD was calculated as per Medbo et al. (1988). RESULTS: AOD values were not different between conditions (p=0.32). Blood pH was lower in the passive (p) recovery compared to active (a) throughout the 2nd and 3rd recovery periods [2nd recovery: 7.18±0.08 to 7.24±0.09 (p), 7.23±0.07to 7.32±0.07 (a), p < 0.05; 3rd recovery: 7.17±0.08 to 7.22±0.09 (p), 7.23±0.08 to 7.32±0.08 (a), p < 0.05]. Average peak VO2 did not vary between conditions for any of the exercise bouts, and exercise performance times (time to volitional fatigue) did not differ between recovery conditions (p=0.28). CONCLUSIONS: Blood acidosis increased during each successive passive recovery period. Subsequent exercise performance, however, was not influenced by the pH perturbation, as AOD, VO2 peak and performance times remained unchanged between conditions.

Generalized Two-Dimensional Heterocorrelation Analysis of Spectrally Resolved and Temporally Resolved Fluorescence of the 8-Anilino-1-naphthalenesulfonate−Apomyoglobin Complex with pH Perturbation

We demonstrate two-dimensional heterocorrelation analysis between spectrally resolved and temporally resolved fluorescence to investigate the decay dynamics of the 8-anilino-1-naphthalenesulfonate- (ANS-) apomyoglobin complex. The dynamic changes of the lifetime components are disclosed across the emission spectrum with an external pH-perturbation. Two different fluorescence lifetime schemes of the ANS-apomyoglobin complex are revealed. From pH 8.5 to 4.5, the transition of protein conformation from the native state to the folding intermediate, a short lifetime component is found to correlate with a short-wavelength emission whose population diminishes with decreasing pH. The lifetime components reflect the excited-state populations of the nascent and the charge-transfer species. From pH 4.2 to 1.0, the transition from the folding intermediate to the acid-unfolded state, the short lifetime is responsible for a long-wavelength emission and the fraction of this component increases when the solution becomes more acidic. In this pH range, the decay components reflect the ground-state populations of microenvironments. The relative decay dynamics across the emission spectrum are revealed without collecting decays at each wavelength. More importantly, these conclusions are reached without the necessity of statistical fitting of the decay data with an a priori decay model.

Recovery from an activity-induced metabolic acidosis in the American alligator, Alligator mississippiensis

The metabolic acidosis resulting from an intense exercise bout is large in crocodilians. Here we studied recovery from this pH perturbation in the American alligator. Metabolic rate, minute ventilation, arterial pH and gases, and strong ion concentration were measured for 10 h after exhaustion to elucidate the mechanisms and time course of recovery. Exhaustion resulted in a significant increase in lactate, metabolic rate, and ventilation, and a decrease in arterial P CO 2 , pH and bicarbonate. By 15 min after exhaustion, oxygen consumption returned to rest though carbon dioxide excretion remained elevated for 30 min. Arterial P O 2 , [Na +], and [K +], increased following exhaustion and recovered by 30 min post-exercise. Minute ventilation, tidal volume, [Cl −], and respiratory exchange ratio returned to resting values by 1 h. The air convection requirement for oxygen was elevated between 15 and 60 min of recovery. Breathing frequency and pH returned to resting values by 2 h of recovery. Lactate levels remained elevated until 6 h post-exercise. Arterial P CO 2 and [HCO 3 −] were depressed until 8 h post-exercise. Compensation during recovery of acid-base balance was achieved by altering ventilation: following the initial metabolic acidosis and titration of bicarbonate, a relative hyperventilation prevented a further decrease in pH.

Thiolate-capped PbS nanocrystals in water: sensitivity to O2, pH and concentration, an alternate pathway for crystal growth and a top-down synthesis

The mechanism of formation of water-soluble, thiolate-capped PbS nanocrystals has been investigated using dynamic light scattering. An evaluation of the potential for use of these species as optical contrast agents in biological imaging applications has also been performed in terms of the responses of the nanocrystals to O2 and to perturbations in pH and concentration. In the synthesis of PbS colloids, addition of sodium sulfide to a mixture of lead acetate, thioglycerol and dithioglycerol in water at high pH gave not only the intended nanocrystals, but also a small concentration of micron-sized particles. Over time, both Ostwald ripening of the nanocrystals and ligand etching of these large particles were at play, with the breakdown of large particles being accelerated by light. Exposure of solutions containing PbS nanocrystals to air resulted in aggregation as a consequence of oxidation of coordinated thiolate to disulfide, but the nanocrystals did not catalyze this process. Lowering the pH of solutions containing the nanocrystals from 11.5 to 8.4 also brought about aggregation through protonation of the surface-bound thiolate ligands. A ten-fold dilution of PbS nanocrystals into H2O at pH 11 resulted in size focusing, but no change in the average size of the particles, whereas a ten-fold dilution into H2O containing excess thiolate at pH 11 caused degradation of the particles into very small clusters. The demonstrated chemical instability of thiolate-capped PbS nanocrystals to acid, O2 and changes in concentration make it unlikely that they will serve as effective near infrared fluorophores in biological imaging applications. A “top-down” protocol for the synthesis of PbS nanocrystals is also reported: bulk PbS was dissolved in aqueous solutions containing thioglycerol and dithioglycerol at high pH to give particles whose mean hydrodynamic diameters could be tuned between 18 and 27 nm by varying the PbS ∶ dithioglycerol ratio.

Utility of deep sea CO2 release experiments in understanding the biology of a high‐CO2 ocean: Effects of hypercapnia on deep sea meiofauna

Oceanic CO2 levels are expected to rise during the next 2 centuries to levels not seen for 10–150 million years by the uptake of atmospheric CO2 in surface waters or potentially through the disposal of waste CO2 in the deep sea. Changes in ocean chemistry caused by CO2 influx may have broad impacts on ocean ecosystems. Physiological processes animals use to cope with CO2‐related stress are known, but the range of sensitivities and effects of changes in ocean chemistry on most ocean life remain unclear. We evaluate the effectiveness of various designs for in situ CO2 release experiments in producing stable perturbations in seawater chemistry over experimental seafloor plots, as is desirable for evaluating the CO2 sensitivities of deep sea animals. We also discuss results from a subset of these experiments on the impacts of hypercapnia on deep sea meiofauna, in the context of experimental designs. Five experiments off central California show that pH perturbations were greatest for experiments using “point source” CO2 pools surrounded by experimental plots. CO2 enclosure experiments with experimental plots positioned within a circular arrangement of CO2 pools had more moderate pH variation. The concentration of dissolution plumes from CO2 pools were related to the speed and turbulence of near‐bottom currents, which influence CO2 dissolution and advection. Survival of meiofauna (nematodes, amoebae, euglenoid flagellates) was low after episodic severe hypercapnia but lower and variable where pH changes ranged from 0 to 0.2 pH units below normal.

Phenotypic and Genotypic Selection of Microbiota Surviving under Dental Restorations

The effects of sealing infected carious dentine below dental restorations on the phenotypic and genotypic diversity of the surviving microbiota was investigated. It was hypothesized that the microbiota would be subject to nutrient limitation or nutrient simplification, as it would no longer have access to dietary components or salivary secretion for growth. The available nutrients would be limited primarily to serum proteins passing from the pulp through the patent dentinal tubules to the infected dentine. Ten lesions were treated, and infected dentine was sealed below dental restorations for approximately 5 months. Duplicate standardized samples of infected dentine were taken at baseline and after the removal of the restorations. The baseline microbiota were composed primarily of Lactobacillus spp., Streptococcus mutans, Streptococcus parasanguinis, Actinomyces israelii, and Actinomyces gerencseriae. None of these taxa were isolated among the microbiota of the dentine samples taken after 5 months, which consisted of only Actinomyces naeslundii, Streptococcus oralis, Streptococcus intermedius, and Streptococcus mitis. The microbiota of the final sample exhibited a significantly (P < 0.001) increased ability to produce glycosidic enzymes (sialidase, beta-N-acetylglucosaminidase, and beta-galactosidase), which liberate sugars from glycoproteins. The genotypic diversity of S. oralis and A. naeslundii was significantly (P = 0.002 and P = 0.001, respectively) reduced in the final samples. There was significantly (P < 0.001) greater genotypic diversity within these taxa between the pairs of dentine samples taken at baseline than was found in the 5-month samples, indicating that the dentine was more homogenous than it was at baseline. We propose that during the interval between placement of the restorations and their removal, the available nutrient, primarily serum proteins, or the relative simplicity and homogeneity of the nutrient supply significantly affected the surviving microbiota. The surviving microbiota was less complex, based on compositional, phenotypic, and genotypic analyses, than that isolated from carious lesions which were also exposed to salivary secretions and pH perturbations.

Phosphoenolpyruvate carboxykinase in cucumber plants is increased both by ammonium and by acidification, and is present in the phloem.

In cucumber ( Cucumis sativus L.), phosphoenolpyruvate carboxykinase (PEPCK) was shown by activity measurements and immunoblots to be present in leaves, stems, roots, flowers, fruit and seed. However, immunolocalisation showed that it was present only in certain cell types. PEPCK was present in the companion cells of the adaxial phloem of minor veins, the adaxial and abaxial phloem of larger veins, the internal and external phloem of vascular bundles in petioles and stems, the phloem in roots and the extra-fascicular phloem in leaves, cotyledons, petioles and stems. Immunohistochemical evidence suggests that both the extra-fascicular phloem and the adaxial phloem are involved in the transport of amino acids. In roots and stems, the abundance of PEPCK was greatly increased by watering plants with a solution of ammonium chloride at low, but not at high pH. PEPCK also increased in leaves, but not roots or stems, of seedlings grown in an atmosphere containing 5% CO(2), and in roots and stems of seedlings watered with butyric acid. All these treatments are known to lower the pH of plant cells. Amino acid metabolism in the phloem may produce an excess of carbon skeletons, pH perturbations and an imbalance in the production/utilisation of NADH. This raises the possibility that PEPCK may function in the conversion of these carbon skeletons to PEP, which, depending on the energy requirements of the phloem, is subsequently utilised by either gluconeogenesis or the Krebs cycle, which both consume protons.

Supramolecular hydrogels respond to ligand-receptor interaction.

N-(Fluorenyl-9-Methoxycarbonyl) dipeptides form supramolecular hydrogels via hydrogen bonding and hydrophobic interactions. These hydrogels respond to a ligand-receptor interaction as well as to thermal or pH perturbation and also exhibit chiral recognition.

A submersible autonomous sensor for spectrophotometric pH measurements of natural waters.

An autonomous sensor for long-term in situ measurements of the pH of natural waters is described. The system is based upon spectrophotometric measurements of a mixture of sample and sulfonephthalein indicator. A simple plumbing design, using a small, low-power solenoid pump and valve, avoids the need for quantitative addition of indicator. A approximately 50-microL slug of indicator is pulled into the sample stream by the pump, and subsequent pumping and mixing results in a section of indicator and sample where absorbance measurements can be made. The design also permits direct determination of the indicator pH perturbation. Absorbances are recorded at three wavelengths (439, 579, and 735 nm) using a custom-built 1.7-cm path length fiber-optic flow cell. Solution blanks are obtained by periodically flushing the cell with sample. Field tests were performed in a local river over an 8-day period. The in situ accuracy, based on comparison with laboratory spectrophotometric pH measurements, was -0.003 pH unit (n = 16), similar to the measurement precision. No drift was observed during the 8-day period. The absorbance ratio used to calculate pH, in combination with a simple and robust optical design, imparts an inherent stability not achievable with conventional potentiometric methods, making the design feasible for long-term autonomous pH measurements.

Renal and systemic pH imaging by contrast-enhanced MRI.

Perturbations of renal and systemic pH accompany diseases of the kidney, such as renal tubular acidosis, and the ability to image tissue pH would be helpful to assess the extent and severity of such conditions. A dual-contrast-agent strategy using two gadolinium agents, the pH-insensitive GdDOTP(5-) and the pH-sensitive GdDOTA-4AmP(5-), has been developed to generate pH maps by MRI. The renal pharmacokinetics of the structurally dissimilar pH-insensitive contrast agents GdDTPA(2-) and GdDOTP(5-) were found to be similar. On that basis, and on the basis of similarity of structure and charge, the renal pharmacokinetics of GdDOTP(5-) and GdDOTA-4AmP(5-) were assumed to be identical. Dynamic T(1)-weighted images of mice were acquired for 1 hr each following boluses of GdDOTP(5-) and GdDOTA-4AmP(5-). The time-varying apparent concentration of GdDOTP(5-) and the time-varying enhancement in longitudinal relaxation rate following GdDOTA-4AmP(5-) were calculated for each pixel and used to compute pH images of the kidneys and surrounding tissues. MRI pH maps of control mice show acidic regions corresponding to the renal papilla, calyx, and ureter. Pretreatment of mice with the carbonic anhydrase inhibitor acetazolamide resulted in systemic metabolic acidosis and accompanying urine alkalinization that was readily detected by this dual-contrast-agent approach.

Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP

BackgroundThe green fluorescent protein (GFP) has been widely used in cell biology as a marker of gene expression, label of cellular structures, fusion tag or as a crucial constituent of genetically encoded biosensors. Mutagenesis of the wildtype gene has yielded a number of improved variants such as EGFP or colour variants suitable for fluorescence resonance energy transfer (FRET). However, folding of some of these mutants is still a problem when targeted to certain organelles or fused to other proteins.ResultsBy directed rational mutagenesis, we have produced a new variant of the Sapphire mutant of GFP with improved folding properties that turns out to be especially beneficial when expressed within organelles or as a fusion tag. Its absorption spectrum is pH-stable and the pKa of its emission is 4.9, making it very resistant to pH perturbation inside cells.Conclusion"T-Sapphire" and its circular permutations can be used as labels of proteins or cellular structures and as FRET donors in combination with red-fluorescent acceptor proteins such as DsRed, making it possible to completely separate donor and acceptor excitation and emission in intensity-based FRET experiments.

Conformational Behavior of Polyelectrolyte Complexes at the Solid/Liquid Interface

Conformation of polymers at solid/liquid interface is an important property that determines their effectiveness in processes such as dispersion, deposition, adhesion, and wettability. In this work, conformational behavior of pyrene labeled poly(maleic acid-co-propylene), MS−P*, in solution and at the alumina surface and its changes upon perturbation in solution pH or addition of a second oppositely charged polymer than the ones in solution were studied using fluorescence spectroscopy. Conformation of this weak polyanion, both in solution and at the solid/liquid interface was found to be strongly pH-dependent. The degree of coiling on alumina depended on the solution state immediately before the adsorption. The conformation of the polymers in solution and on the solid were the same above pH 4, but, interestingly, the adsorbed polymer was more stretched at low pH. When the polymer is in the coiled form, changes in pH caused partial conformational adjustments of the adsorbed species. However, in the stretched form, the adsorbed polymer was unable to adopt the coiled conformation on the surface even with pH perturbation. Addition of polycations, poly(ethyleneimine) and poly(diallyldimethylammonium) chloride, to systems containing the preadsorbed polyanion led to conformational changes due to the formation of complexes between them at the surface. While the polyelectrolyte adsorbed in the stretched form undergoes very little conformational rearrangement, the re-conformation of the polymer adsorbed in the coiled form depends on the charge density of the second polymer and the solid. These results are compared with the behavior of polyelectrolyte complexes in solution and analyzed to elucidate possible conformational dynamics of polymers at interfaces.

The response of GS-NS0 myeloma cells to single and multiple pH perturbations.

Animal cells are cultured in several types of vessels at laboratory and industrial scale the most common being the stirred tank and the air-lift. Economically, it is preferable to culture animal cells at the largest possible scale but the perceived sensitivity of animal cells to hydrodynamic shear has, until now, limited the aeration and agitation rates used. This has been reported to cause inhomogeneities in operational parameters such as dissolved oxygen concentration, temperature and pH. pH is of special interest during the latter stages of many animal cell fermentation because alkali additions, used for pH control, can cause large local pH perturbations of varying size and duration. The effect of single and multiple pH perturbations on the cell growth of a widely used GS-NS0 mouse myeloma cell line grown in batch culture was investigated. The effect of perturbation amplitude and duration was investigated using a single stirred tank reactor (STR). In the single STR system cells were subjected to one pH 8.0 or 9.0 perturbation ranging in duration from 0-90 minutes. No measurable decrease in viable cell number was seen for pH 8.0 perturbations of any duration whereas pH 9.0 perturbations lasting for 10 minutes caused a 15% decrease in viable cell number. The proportion of viable cells decreased with increasing perturbation time and a 90-minute exposure killed all of the cells. The effect of multiple pH perturbations on GS-NS0 cells was investigated using two connected STR's. More specifically the number of perturbations and the perturbation frequency were investigated. Cells were subjected to between 0 and 100 perturbations at pH 8.0; the time between each perturbation (frequency) was 6 minutes and each perturbation lasted for 200 seconds. Viable cell number decreased with increasing perturbation number, with 100 perturbations causing death of 27.5% of cells. Cells were also exposed to 10 perturbations at pH 9.0, each of 200 second duration at frequencies of either 6, 18 or 60 minutes. Approximately 8 times more cells were killed with perturbations at a 6-minute frequency (28.3% cell death) than at a 60-minute frequency (3.4% cell death).

Spectrophotometric pH measurements of freshwater

The use of cresol red (CR) indicator for determination of freshwater pH is evaluated. Ionic strength effects and indicator pH perturbation are discussed and quantified using theoretical and empirical approaches. Spectrophotometric and potentiometric methods are directly compared by repeated analyses of a low ionic strength pH buffer. The mean and standard deviation of the two methods were 7.618±0.008 (spectrophotometric) and 7.484±0.040 (potentiometric) (N=18) with systematic errors of 0.003 and 0.137 pH units relative to the true pH (7.621). Field data from an alkaline river (pH∼7.8–8.8) show that measurement reproducibility is better than 0.01 pH units, making it possible to resolve very small spatial and temporal changes in riverine pH. Uncertainty in the indicator apparent dissociation constant limits the accuracy of the pH measurement to ∼0.05 pH units. An alternative method for estimating the dissociation constant, based on calculation of pH from two other carbonate parameters, is proposed.

Carbon cycle. Potential impacts of CO2 injection on deep-sea biota.

Efforts to reduce carbon dioxide emissions are increasingly looking to the oceans, either through iron fertilization programs (see this week9s Policy Forum by Chisholm et al.) or through COinjection into the deep sea. In their Perspective, Seibel and Walsh investigate how such deep-sea disposal may affect organisms that live in these environments. They warn that even small perturbations in COor pH may have important consequences for deep-sea ecosystems and for global biogeochemical cycles. Detailed studies into these effects are needed before the risks and benefits of deep-sea carbon storage can be assessed appropriately.

The response of GS‐NS0 myeloma cells to pH shifts and pH perturbations

The perceived sensitivity of animal cells to hydrodynamic shear has limited agitation and aeration at large-scale. This makes it difficult to ensure adequate mixing of the vessel contents and may lead to inhomogeneities in operational parameters such as temperature, dissolved oxygen concentration, and especially pH. The effect of pH shifts and pH perturbations on the cellular responses, in batch culture, of a GS-NS0 mouse myeloma cell line, expressing a recombinant antibody, was investigated. In addition, the effect of extreme pH on the structure of the purified antibody product was studied using isoelectric focusing. The fermentation pH value was shifted abruptly from pH 7.3 to pH values ranging from 6.5 to 9.0. Culture pH was maintained at this new value for the remainder of the fermentation. All pH shifts of above 0.2 units caused a transient increase in apoptosis. However, cultures shifted to pH values between 7.0 and 8.0 continued to grow and the apoptotic fraction returned to initial levels. Cultures shifted to pH values above pH 8.0 and below pH 7.0 did not recover resulting in culture death. For example, a shift to pH 8.5 caused accumulation of cells in the G(2)/M phase of the cell cycle followed by apoptotic death. After the pH shift, maximum specific growth rate was observed over the range pH 7.3 to 7.5 and maximum viable cell number was seen at pH 7.3. Maximum volumetric antibody production, resulting from increased culture longevity, was seen at pH 7.0. It was also observed that glucose consumption increased with increasing pH. In a separate set of experiments cells were subjected to a single pH perturbation ranging in duration from 0 to 600 minutes. Exposure of cells to a pH value greater than 8.5 for more than 10 minutes caused a decrease in the proportion of viable cells and induced a lag in cell growth. At very low pH (6.5) similar effects were seen, but only for extended perturbations (600 min). However, after recovery from the pH perturbation, growth, product secretion and metabolism all returned to original levels. Incubation of the antibody, at the range of pH values investigated, indicated no alterations in the structure of the antibody as determined by the isoelectric focusing pattern.

Compensatory Dynamics in Planktonic Community Responses to pH Perturbations

Compensatory Dynamics in Planktonic Community Responses to pH Perturbations

COMPENSATORY DYNAMICS IN PLANKTONIC COMMUNITY RESPONSES TO pH PERTURBATIONS

Compensatory population dynamics, in which species that decline in response to an environmental perturbation are replaced by similar species, may be crucial in maintaining processes performed by functional groups of species. Compensatory dynamics may be produced by negative interactions among species, such that the decrease in abundance of a species releases the suppression of another species and allows it to increase. We conducted a mesocosm experiment in Trout Lake, Wisconsin, USA, to test the hypothesis that compensatory shifts in species abundances play a role in overall planktonic community response to pH perturbation. In 2000-L mesocosms over a period of six weeks, we contrasted a control treatment with two acidified treatments (press, sustained pH = 4.7; and pulse, alternating pH = 4.7 and ambient pH). In the acidified treatments, we saw changes in abundance of the major zooplankton and phytoplankton species, but we observed few cases of compensatory dynamics. Nonetheless, when present, compensatory dynamics could be strong. Analyses using autoregressive models revealed negative interactions among species that could potentially lead to compensatory dynamics. However, this potential for compensatory dynamics was not realized in cases where all species were sensitive to the pH perturbations. Therefore, compensatory dynamics that buffer community responses to perturbations may be limited in communities in which many species are sensitive to the perturbation.

GABAergic excitation and K+-mediated volume transmission in the hippocampus

Publisher Summary The mechanisms underlying the GABA-mediated depolarizing non-synaptic potential (GDNSP) is discussed in this chapter. In particular, chapter provides evidence that the GDNSP is largely due to an activity-induced potassium transient, which has a direct depolarizing effect even in the absence of glutamatergic transmission. This transient increase in the extracellular K + concentration seems to be tightly linked to the GABA A receptor-dependent coupling in the local interneuronal network that is functionally excitatory in the presence, but not in the absence, of CO 2 and HCO 3 . In general, it appears that bicarbonate-dependent volume transmission mediated by K + plays a salient role in GABAergic neuronal responses during synchronous high-frequency activity. Another type of ionic shift that is triggered by tonic GABAergic activity is an intraneuronal acidicosis coupled to an extracellular alkaline pH change, which are direct consequences of the GABA A receptor-mediated net efflux of HCO 3 . In view of the large diversity of both ligand-gated and voltage-gated channels, which are sensitive to perturbations of extracellular pH, both H + and HCO 3 - transients generated in the interstitial space are likely to have a volume signaling role of their own.

16S rDNA-based characterization of BTX-catabolizing microbial associations isolated from a South African sandy soil.

In the presence of different selection pressures, particularly pH and electron donor concentration, indigenous microbial associations which catabolize selected petroleum hydrocarbon components (benzene, toluene and o-, m- and p-xylene (BTX)) were enriched and isolated from a petroleum hydrocarbon-contaminated KwaZulu-Natal sandy soil. Electron microscopy revealed that, numerically, rods constituted the majority of the populations responsible for BTX catabolism. Molecular techniques (polymerase chain reaction (PCR) and 16S rDNA fingerprinting by denaturing-gradient gel electrophoresis (DGGE)) were employed to explore the diversities and analyze the structures of the isolated microbial associations. Pearson product-moment correlation indicated that the different, but chemically similar, petroleum hydrocarbon molecules, effected the isolation of different associations. However, some similar numerically-dominant bands characterized the associations. A 30% similarity was evident between the m- and o-xylene-catabolizing associations regardless of the molecule concentration and the enrichment pH. PCR-DGGE was also used to complement conventional culture-based microbiological procedures for environmental parameter optimization. Band pattern differences indicated profile variations of the isolated associations which possibly accounted for the growth rate changes recorded in response to pH and temperature perturbations.