Intracellular pH in rat pancreatic ducts

DEFF Research Database (Denmark)

Novak, I; Hug, M; Greger, R

1997-01-01

In order to study the mechanism of H+ and HCO3- transport in a HCO3- secreting epithelium, pancreatic ducts, we have measured the intracellular pH (pHi) in this tissue using the pH sensitive probe BCECF. We found that exposures of ducts to solutions containing acetate/acetic acid or NH4+/NH3...... buffers (20 mmol/l) led to pHi changes in accordance with entry of lipid-soluble forms of the buffers, followed by back-regulation of pHi by duct cells. In another type of experiment, changes in extracellular pH of solutions containing HEPES or HCO3-/CO2 buffers led to significant changes in pHi that did....... Under some conditions, these exchangers can be invoked to regulate cell pH....

Imaging of Intracellular pH in Tumor Spheroids Using Genetically Encoded Sensor SypHer2.

Science.gov (United States)

Zagaynova, Elena V; Druzhkova, Irina N; Mishina, Natalia M; Ignatova, Nadezhda I; Dudenkova, Varvara V; Shirmanova, Marina V

2017-01-01

Intracellular pH (pHi) is one of the most important parameters that regulate the physiological state of cells and tissues. pHi homeostasis is crucial for normal cell functioning. Cancer cells are characterized by having a higher (neutral to slightly alkaline) pHi and lower (acidic) extracellular pH (pHe) compared to normal cells. This is referred to as a "reversed" pH gradient, and is essential in supporting their accelerated growth rate, invasion and migration, and in suppressing anti-tumor immunity, the promotion of metabolic coupling with fibroblasts and in preventing apoptosis. Moreover, abnormal pH, both pHi and pHe, contribute to drug resistance in cancers. Therefore, the development of methods for measuring pH in living tumor cells is likely to lead to better understanding of tumor biology and to open new ways for cancer treatment. Genetically encoded, fluorescent, pH-sensitive probes represent promising instruments enabling the subcellular measurement of pHi with unrivaled specificity and high accuracy. Here, we describe a protocol for pHi imaging at a microscopic level in HeLa tumor spheroids, using the genetically encoded ratiometric (dual-excitation) pHi indicator, SypHer2.

Intracellular pH is a tightly controlled signal in yeast

NARCIS (Netherlands)

Orij, R.; Brul, S.; Smits, G.J.

2011-01-01

Background: Nearly all processes in living cells are pH dependent, which is why intracellular pH (pHi) is a tightly regulated physiological parameter in all cellular systems. However, in microbes such as yeast, pHi responds to extracellular conditions such as the availability of nutrients. This

KCC2-dependent Steady-state Intracellular Chloride Concentration and pH in Cortical Layer 2/3 Neurons of Anesthetized and Awake Mice.

Science.gov (United States)

Boffi, Juan C; Knabbe, Johannes; Kaiser, Michaela; Kuner, Thomas

2018-01-01

Neuronal intracellular Cl - concentration ([Cl - ] i ) influences a wide range of processes such as neuronal inhibition, membrane potential dynamics, intracellular pH (pH i ) or cell volume. Up to date, neuronal [Cl - ] i has predominantly been studied in model systems of reduced complexity. Here, we implemented the genetically encoded ratiometric Cl - indicator Superclomeleon (SCLM) to estimate the steady-state [Cl - ] i in cortical neurons from anesthetized and awake mice using 2-photon microscopy. Additionally, we implemented superecliptic pHluorin (SE-pHluorin) as a ratiometric sensor to estimate the intracellular steady-state pH (pH i ) of mouse cortical neurons in vivo . We estimated an average resting [Cl - ] i of 6 ± 2 mM with no evidence of subcellular gradients in the proximal somato-dendritic domain and an average somatic pH i of 7.1 ± 0.2. Neither [Cl - ] i nor pH i were affected by isoflurane anesthesia. We deleted the cation-Cl - co-transporter KCC2 in single identified neurons of adult mice and found an increase of [Cl - ] i to approximately 26 ± 8 mM, demonstrating that under in vivo conditions KCC2 produces low [Cl - ] i in adult mouse neurons. In summary, neurons of the brain of awake adult mice exhibit a low and evenly distributed [Cl - ] i in the proximal somato-dendritic compartment that is independent of anesthesia and requires KCC2 expression for its maintenance.

Regulation of intracellular pH in cnidarians: response to acidosis in Anemonia viridis.

Science.gov (United States)

Laurent, Julien; Venn, Alexander; Tambutté, Éric; Ganot, Philippe; Allemand, Denis; Tambutté, Sylvie

2014-02-01

The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO₂-driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH₄Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na⁺-free seawater indicate a potential role of Na⁺/H⁺ plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited. © 2013 FEBS.

Microscopic monitoring of extracellular pH in dental biofilms

DEFF Research Database (Denmark)

Schlafer, Sebastian; Garcia, Javier; Greve, Matilde

pH in dental biofilm is a key virulence factor for the development of caries lesions. The complex three-dimensional architecture of dental biofilms leads to steep gradients of nutrients and metabolites, including organic acids, across the biofilm. For decades, measuring pH in dental biofilm has...... been limited to monitoring bulk pH with electrodes. Although pH microelectrodes with a better spatial resolution have been developed, they do not permit to monitor horizontal pH gradients in real-time. Quantitative fluorescent microscopic techniques, such as fluorescence lifetime imaging or pH...... ratiometry, can be employed to map the pH landscape in dental biofilm with more detail. However, when pH sensitive fluorescent probes are used to visualize pH in biofilms, it is crucial to differentiate between extracellular and intracellular pH. Intracellular microbial pH and pH in the extracellular matrix...

Intracellular pH and inorganic phosphate content of heart in vivo: A 31P-NMR study

International Nuclear Information System (INIS)

Katz, L.A.; Swain, J.A.; Portman, M.A.; Balaban, R.S.

1988-01-01

Studies were performed to determine the contribution of red blood cells to the 31 P-nuclear magnetic resonance (NMR) spectrum of the canine heart in vivo and the feasibility of measuring myocardial intracellular phosphate and pH. This was accomplished by replacing whole blood with a perfluorochemical perfusion emulsion blood substitute, Oxypherol, and noting the difference in the 31 P-NMR spectrum of the heart. NMR data were collected with a NMR transmitter-receiver coil on the surface of the distal portion of the left ventricle. These studies demonstrated that a small contribution from 2,3-diphosphoglycerate (2,3-DPG) and phosphodiesters in the blood could be detected. The magnitude and shift of these blood-borne signals permitted the relative quantification of intracellular inorganic phosphate (P i ) content as well as intracellular pH. Under resting conditions, the intracellular ATP/P i was 7.0 ± 0.08. This corresponds to a free intracellular P 1 content of ∼ 0.8 μmol./g wet wt. The intracellular pH was 7.10 ± 0.01. Acute respiratory alkalosis and acidosis, with the arterial pH ranging from ∼7.0 to 7.7, resulted in only small changes in the intracellular pH. These latter results demonstrate an effective myocardial intracellular proton-buffering mechanism in vivo

Preparation and evaluation of Vinpocetine self-emulsifying pH gradient release pellets.

Science.gov (United States)

Liu, Mengqi; Zhang, Shiming; Cui, Shuxia; Chen, Fen; Jia, Lianqun; Wang, Shu; Gai, Xiumei; Li, Pingfei; Yang, Feifei; Pan, Weisan; Yang, Xinggang

2017-11-01

The main objective of this study was to develop a pH gradient release pellet with self-emulsifying drug delivery system (SEDDS), which could not only improve the oral bioavailability of Vinpocetine (VIN), a poor soluble drug, but reduce the fluctuation of plasma concentration. First, the liquid VIN SEDDS formulation was prepared. Then the self-emulsifying pH gradient release pellets were prepared by extrusion spheronization technique, and formulation consisted by the liquid SEDDS, absorbent (colloidal silicon dioxide), penetration enhancer (sodium chloride), microcrystalline cellulose, ethyl alcohol, and three coating materials (HPMC, Eudragit L30D55, Eudragit FS30D) were eventually selected. Three kinds of coated pellets were mixed in capsules with the mass ratio of 1:1:1. The release curves of capsules were investigated in vitro under the simulated gastrointestinal conditions. In addition, the oral bioavailability and pharmacokinetics of VIN self-emulsifying pH gradient release pellets, commercial tablets and liquid VIN SEDDS were evaluated in Beagle dogs. The oral bioavailability of self-emulsifying pH gradient release pellets was about 149.8% of commercial VIN tablets, and it was about 86% of liquid VIN SEDDS, but there were no significant difference between liquid SEDDS and self-emulsifying pH gradient release pellets. In conclusion, the self-emulsifying pH gradient release pellets could significantly enhance the absorption of VIN and effectively achieve a pH gradient release. And the self-emulsifying pH gradient release pellet was a promising method to improve bioavailability of insoluble drugs.

Intracellular pH distribution as a cell health indicator in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Aabo, Thomas; Glückstad, Jesper; Siegumfeldt, Henrik

2011-01-01

.d.(pHint)) to describe the internal pH distributions. The cellular pH distributional response to external stress such as heat has not previously been determined. In this study, the intracellular pH (pHi) and the s.d.(pHint) of Saccharomyces cerevisiae cells exposed to supralethal temperatures were measured using...

A novel FbFP-based biosensor toolbox for sensitive in vivo determination of intracellular pH.

Science.gov (United States)

Rupprecht, Christian; Wingen, Marcus; Potzkei, Janko; Gensch, Thomas; Jaeger, Karl-Erich; Drepper, Thomas

2017-09-20

The intracellular pH is an important modulator of various bio(techno)logical processes such as enzymatic conversion of metabolites or transport across the cell membrane. Changes of intracellular pH due to altered proton distribution can thus cause dysfunction of cellular processes. Consequently, accurate monitoring of intracellular pH allows elucidating the pH-dependency of (patho)physiological and biotechnological processes. In this context, genetically encoded biosensors represent a powerful tool to determine intracellular pH values non-invasively and with high spatiotemporal resolution. We have constructed a toolbox of novel genetically encoded FRET-based pH biosensors (named Fluorescence Biosensors for pH or FluBpH) that utilizes the FMN-binding fluorescent protein EcFbFP as donor domain. In contrast to many fluorescent proteins of the GFP family, EcFbFP exhibits a remarkable tolerance towards acidic pH (pK a ∼3.2). To cover the broad range of physiologically relevant pH values, three EYFP variants exhibiting pK a values of 5.7, 6.1 and 7.5 were used as pH-sensing FRET acceptor domains. The resulting biosensors FluBpH 5.7, FluBpH 6.1 and FluBpH 7.5 were calibrated in vitro and in vivo to accurately evaluate their pH indicator properties. To demonstrate the in vivo applicability of FluBpH, changes of intracellular pH were ratiometrically measured in E. coli cells during acid stress. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluating Nanoparticle Sensor Design for Intracellular pH Measurements

DEFF Research Database (Denmark)

Benjaminsen, Rikke Vicki; Sun, Honghao; Henriksen, Jonas Rosager

2011-01-01

Particle-based nanosensors have over the last decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors is challenging...... and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle...... quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pKa to each sensor, seem to be a solution...

Systematic generation of buffer systems for pH gradient ion exchange chromatography and their application.

Science.gov (United States)

Kröner, Frieder; Hubbuch, Jürgen

2013-04-12

pH gradient protein separations are widely used techniques in the field of protein analytics, of which isoelectric focusing is the most well known application. The chromatographic variant, based on the formation of pH gradients in ion exchange columns is only rarely applied due to the difficulties to form controllable, linear pH gradients over a broad pH range. This work describes a method for the systematic generation of buffer compositions with linear titration curves, resulting in well controllable pH gradients. To generate buffer compositions with linear titration curves an in silico method was successfully developed. With this tool, buffer compositions for pH gradient ion exchange chromatography with pH ranges spanning up to 7.5 pH units were established and successfully validated. Subsequently, the buffer systems were used to characterize the elution behavior of 22 different model proteins in cation and anion exchange pH gradient chromatography. The results of both chromatographic modes as well as isoelectric focusing were compared to describe differences in between the methods. Copyright © 2013 Elsevier B.V. All rights reserved.

Intracellular pH in sperm physiology.

Science.gov (United States)

Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

2014-08-01

Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

The effects of extracellular pH and hydroxycinnamic acids influence the intracellular pH of Brettanomyces bruxellensis DSM 7001

DEFF Research Database (Denmark)

Campolongo, Simona; Siegumfeldt, Henrik; Aabo, Thomas Ask

2014-01-01

and intracellular pH changes in B. bruxellensis DSM 7001, in response to extracellular pH, as well as to the presence of an energy source and hydroxycinnamic acids, have been investigated in this paper by means of Fluorescent Ratio Imaging Microscopy (FRIM). The results show that B. bruxellensis DSM 7001 is able...

Evaluating nanoparticle sensor design for intracellular pH measurements.

Science.gov (United States)

Benjaminsen, Rikke V; Sun, Honghao; Henriksen, Jonas R; Christensen, Nynne M; Almdal, Kristoffer; Andresen, Thomas L

2011-07-26

Particle-based nanosensors have over the past decade been designed for optical fluorescent-based ratiometric measurements of pH in living cells. However, quantitative and time-resolved intracellular measurements of pH in endosomes and lysosomes using particle nanosensors are challenging, and there is a need to improve measurement methodology. In the present paper, we have successfully carried out time-resolved pH measurements in endosomes and lyosomes in living cells using nanoparticle sensors and show the importance of sensor choice for successful quantification. We have studied two nanoparticle-based sensor systems that are internalized by endocytosis and elucidated important factors in nanosensor design that should be considered in future development of new sensors. From our experiments it is clear that it is highly important to use sensors that have a broad measurement range, as erroneous quantification of pH is an unfortunate result when measuring pH too close to the limit of the sensitive range of the sensors. Triple-labeled nanosensors with a pH measurement range of 3.2-7.0, which was synthesized by adding two pH-sensitive fluorophores with different pK(a) to each sensor, seem to be a solution to some of the earlier problems found when measuring pH in the endosome-lysosome pathway.

A Gold Nanoparticle Bio-Optical Transponder to Dynamically Monitor Intracellular pH.

Science.gov (United States)

Carnevale, Kate J F; Riskowski, Ryan A; Strouse, Geoffrey F

2018-06-13

A pH-sensitive bio-optical transponder (pH-BOT) capable of simultaneously reporting the timing of intracellular DNA cargo release from a gold nanoparticle (AuNP) and the evolving intracellular pH (pH i) during endosomal maturation is demonstrated. The pH-BOT is designed with a triple-dye-labeled duplex DNA appended to a 6.6 nm AuNP, utilizing pH-responsive fluorescein paired with DyLight405 as a surface energy transfer (SET) coupled dye pair to ratiometrically report the pH at and after cargo release. A non-SET-coupled dye, DyLight 700, is used to provide dynamic tracking throughout the experiment. The pH-BOT beacon of the cargo uptake, release, and processing was visualized using live-cell confocal fluorescent microscopy in Chinese hamster ovary cells, and it was observed that while maturation of endosomes carrying pH-BOT is slowed significantly, the pH-BOT is distributed throughout the endolysosomal system while remaining at pH ∼6. This observed decoupling of endosomal maturation from acidification lends support to those models that propose that pH alone is not sufficient to explain endosomal maturation and may enable greater insight into our understanding of the fundamental processes of biology.

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum.

Science.gov (United States)

da Rosa-Garzon, Nathália Gonsales; Laure, Hélen Julie; Souza-Motta, Cristina Maria de; Rosa, José César; Cabral, Hamilton

2017-08-09

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96 hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral-alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.

Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells.

Science.gov (United States)

Chen, Gunng-Shinng; Lee, Shiao-Pieng; Huang, Shu-Fu; Chao, Shih-Chi; Chang, Chung-Yi; Wu, Gwo-Jang; Li, Chung-Hsing; Loh, Shih-Hurng

2018-06-01

Homeostasis of intracellular pH (pH i ) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na + -H + exchanger (NHE), Na + -HCO 3 - co-transporter (NBC), Cl - /HCO 3 - exchanger (AE) and Cl - /OH - exchanger (CHE) have been identified to co-regulate pH i homeostasis. However, functional and biological pH i -regulators in human dental pulp stem cells (hDPSCs) have yet to be identified. Microspectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pH i changes. NH 4 Cl and Na + -acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pH i -regulators were detected by Western blot technique. The resting pH i was no significant difference between that in HEPES-buffered (nominal HCO 3 - -free) solution or CO 2 /HCO 3 -buffered system (7.42 and 7.46, respectively). The pH i recovery following the induced-intracellular acidosis was blocked completely by removing [Na + ] o , while only slowed (-63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pH i recovery was inhibited entirely by removing [Na + ] o , while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (-55%) the acid extrusion. Both in HEPES-buffered and CO 2 /HCO 3 -buffered system solution, the pH i recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl - ] o . Western blot analysis showed the isoforms of pH i regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs. We demonstrate for the first time that resting pH i is significantly higher than 7.2 and meditates functionally by two Na + -dependent acid extruders (NHE and NBC), two Cl - -dependent acid loaders (CHE and AE) and one Na + -independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry. Copyright © 2018 Elsevier Ltd. All rights reserved.

Modulation of Connexin-36 Gap Junction Channels by Intracellular pH and Magnesium Ions.

Science.gov (United States)

Rimkute, Lina; Kraujalis, Tadas; Snipas, Mindaugas; Palacios-Prado, Nicolas; Jotautis, Vaidas; Skeberdis, Vytenis A; Bukauskas, Feliksas F

2018-01-01

Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pH i ) and cytosolic magnesium ion concentration ([Mg 2+ ] i ), which can vary significantly under various physiological and pathological conditions. However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pH i and [Mg 2+ ] i affect junctional conductance (g j ) in an interdependent manner; in other words, intracellular acidification cause increase or decay in g j depending on whether [Mg 2+ ] i is high or low, respectively, and intracellular alkalization cause reduction in g j independently of [Mg 2+ ] i . Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pH i and [Mg 2+ ] i . Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pH i and [Mg 2+ ] i . Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg 2+ ] i , while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36 * E8Q lost the initial increase of g j at low [Mg 2+ ] i and double mutation lost the sensitivity to high [Mg 2+ ] i . These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg 2+ and H + ions.

Nanoparticle-based luminescent probes for intracellular sensing and imaging of pH.

Science.gov (United States)

Schäferling, Michael

2016-05-01

Fluorescence imaging microscopy is an essential tool in biomedical research. Meanwhile, various fluorescent probes are available for the staining of cells, cell membranes, and organelles. Though, to monitor intracellular processes and dysfunctions, probes that respond to ubiquitous chemical parameters determining the cellular function such as pH, pO2 , and Ca(2+) are required. This review is focused on the progress in the design, fabrication, and application of photoluminescent nanoprobes for sensing and imaging of pH in living cells. The advantages of using nanoprobes carrying fluorescent pH indicators compared to single molecule probes are discussed as well as their limitations due to the mostly lysosomal uptake by cells. Particular attention is paid to ratiometric dual wavelength nanosensors that enable intrinsic referenced measurements. Referencing and proper calibration procedures are basic prerequisites to carry out reliable quantitative pH determinations in complex samples such as living cells. A variety of examples will be presented that highlight the diverseness of nanocarrier materials (polymers, micelles, silica, quantum dots, carbon dots, gold, photon upconversion nanocrystals, or bacteriophages), fluorescent pH indicators for the weak acidic range, and referenced sensing mechanisms, that have been applied intracellularly up to now. WIREs Nanomed Nanobiotechnol 2016, 8:378-413. doi: 10.1002/wnan.1366 For further resources related to this article, please visit the WIREs website. © 2015 Wiley Periodicals, Inc.

Ratiometric photoluminescence sensing based on Ti3C2 MXene quantum dots as an intracellular pH sensor.

Science.gov (United States)

Chen, Xu; Sun, Xueke; Xu, Wen; Pan, Gencai; Zhou, Donglei; Zhu, Jinyang; Wang, He; Bai, Xue; Dong, Biao; Song, Hongwei

2018-01-18

Intracellular pH sensing is of importance and can be used as an indicator for monitoring the evolution of various diseases and the health of cells. Here, we developed a new class of surface-functionalized MXene quantum dots (QDs), Ti 3 C 2 , by the sonication cutting and hydrothermal approach and further explored their intracellular pH sensing. The functionalized Ti 3 C 2 QDs exhibit bright excitation-dependent blue photoluminescence (PL) originating from the size effect and surface defects. Meanwhile, Ti 3 C 2 QDs demonstrate a high PL response induced by the deprotonation of the surface defects. Furthermore, combining the highly pH sensitive Ti 3 C 2 QDs with the pH insensitive [Ru(dpp) 3 ]Cl 2 , we developed a ratiometric pH sensor to quantitatively monitor the intracellular pH values. These novel MXene quantum dots can serve as a promising platform for developing practical fluorescent nanosensors.

Intracellular pH Campylobacter jejuni when treated with aqueous chlorine dioxide

DEFF Research Database (Denmark)

Smigic, Nada; Rajkovic, Andreja; Arneborg, Nils

2011-01-01

The aim of this study was to investigate the response of Campylobacter jejuni at single-cell level when exposed to different concentrations of chlorine dioxide (ClO2). The parameter of choice, intracellular pH (pHi), was determined by using fluorescence ratio imaging microscopy with a p...

Computer simulation of immobilized pH gradients at acidic and alkaline extremes - A quest for extended pH intervals

Science.gov (United States)

Mosher, Richard A.; Bier, Milan; Righetti, Pier Giorgio

1986-01-01

Computer simulations of the concentration profiles of simple biprotic ampholytes with Delta pKs 1, 2, and 3, on immobilized pH gradients (IPG) at extreme pH values (pH 3-4 and pH 10-11) show markedly skewed steady-state profiles with increasing kurtosis at higher Delta pK values. Across neutrality, all the peaks are symmetric irrespective of their Delta pK values, but they show very high contribution to the conductivity of the background gel and significant alteration of the local buffering capacity. The problems of skewness, due to the exponential conductivity profiles at low and high pHs, and of gel burning due to a strong electroosmotic flow generated by the net charges in the gel matrix, also at low and high pHs, are solved by incorporating in the IPG gel a strong viscosity gradient. This is generated by a gradient of linear polyacrylamide which is trapped in the gel by the polymerization process.

BSA-coated nanoparticles for improved SERS-based intracellular pH sensing.

Science.gov (United States)

Zheng, Xiao-Shan; Hu, Pei; Cui, Yan; Zong, Cheng; Feng, Jia-Min; Wang, Xin; Ren, Bin

2014-12-16

Local microenvironment pH sensing is one of the key parameters for the understanding of many biological processes. As a noninvasive and high sensitive technique, surface-enhanced Raman spectroscopy (SERS) has attracted considerable interest in the detection of the local pH of live cells. We herein develop a facile way to prepare Au-(4-MPy)-BSA (AMB) pH nanosensor. The 4-MPy (4-mercaptopyridine) was used as the pH sensing molecule. The modification of the nanoparticles with BSA not only provides a high sensitive response to pH changes ranging from pH 4.0 to 9.0 but also exhibits a high sensitivity and good biocompatibility, stability, and reliability in various solutions (including the solutions of high ionic strength or with complex composition such as the cell culture medium), both in the aggregation state or after long-term storage. The AMB pH nanosensor shows great advantages for reliable intracellular pH analysis and has been successfully used to monitor the pH distribution of live cells and can address the grand challenges in SERS-based pH sensing for practical biological applications.

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

Science.gov (United States)

Zuo, Wu-Lin; Li, Sheng; Huang, Jie-Hong; Yang, Deng-Liang; Zhang, Geng; Chen, Si-Liang; Ruan, Ye-Chun; Ye, Ke-Nan; Cheng, Christopher H K; Zhou, Wen-Liang

2011-01-01

The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+)/HCO(3)(-) cotransporter in the pH regulation in rat epididymis. Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F) and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH) solution, the intracellular pH (pHi) recovery from NH(4)Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+)/H(+) exchanger (NHE). Immediately changing of the KH solution from HEPES buffered to HCO(3)(-) buffered would cause another pHi recovery. The pHi recovery in HCO(3)(-) buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), the inhibitor of HCO(3)(-) transporter or by removal of extracellular Na(+). The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH. The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

A tunable ratiometric pH sensor based on carbon nanodots for the quantitative measurement of the intracellular pH of whole cells.

Science.gov (United States)

Shi, Wen; Li, Xiaohua; Ma, Huimin

2012-06-25

The whole picture: Carbon nanodots labeled with two fluorescent dyes have been developed as a tunable ratiometric pH sensor to measure intracellular pH. The nanosensor shows good biocompatibility and cellular dispersibility. Quantitative determinations on intact HeLa cells and pH fluctuations associated with oxidative stress were performed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Imaging intracellular pH in a reef coral and symbiotic anemone.

Science.gov (United States)

Venn, A A; Tambutté, E; Lotto, S; Zoccola, D; Allemand, D; Tambutté, S

2009-09-29

The challenges corals and symbiotic cnidarians face from global environmental change brings new urgency to understanding fundamental elements of their physiology. Intracellular pH (pHi) influences almost all aspects of cellular physiology but has never been described in anthozoans or symbiotic cnidarians, despite its pivotal role in carbon concentration for photosynthesis and calcification. Using confocal microscopy and the pH sensitive probe carboxy SNARF-1, we mapped pHi in short-term light and dark-incubated cells of the reef coral Stylophora pistillata and the symbiotic anemone Anemonia viridis. In all cells isolated from both species, pHi was markedly lower than the surrounding seawater pH of 8.1. In cells that contained symbiotic algae, mean values of pHi were significantly higher in light treated cells than dark treated cells (7.41 +/- 0.22 versus 7.13 +/- 0.24 for S. pistillata; and 7.29 +/- 0.15 versus 7.01 +/- 0.27 for A. viridis). In contrast, there was no significant difference in pHi in light and dark treated cells without algal symbionts. Close inspection of the interface between host cytoplasm and algal symbionts revealed a distinct area of lower pH adjacent to the symbionts in both light and dark treated cells, possibly associated with the symbiosome membrane complex. These findings are significant developments for the elucidation of models of inorganic carbon transport for photosynthesis and calcification and also provide a cell imaging procedure for future investigations into how pHi and other fundamental intracellular parameters in corals respond to changes in the external environment such as reductions in seawater pH.

KCC2-dependent Steady-state Intracellular Chloride Concentration and pH in Cortical Layer 2/3 Neurons of Anesthetized and Awake Mice

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Juan C. Boffi

2018-01-01

Full Text Available Neuronal intracellular Cl− concentration ([Cl−]i influences a wide range of processes such as neuronal inhibition, membrane potential dynamics, intracellular pH (pHi or cell volume. Up to date, neuronal [Cl−]i has predominantly been studied in model systems of reduced complexity. Here, we implemented the genetically encoded ratiometric Cl− indicator Superclomeleon (SCLM to estimate the steady-state [Cl−]i in cortical neurons from anesthetized and awake mice using 2-photon microscopy. Additionally, we implemented superecliptic pHluorin (SE-pHluorin as a ratiometric sensor to estimate the intracellular steady-state pH (pHi of mouse cortical neurons in vivo. We estimated an average resting [Cl−]i of 6 ± 2 mM with no evidence of subcellular gradients in the proximal somato-dendritic domain and an average somatic pHi of 7.1 ± 0.2. Neither [Cl−]i nor pHi were affected by isoflurane anesthesia. We deleted the cation-Cl− co-transporter KCC2 in single identified neurons of adult mice and found an increase of [Cl−]i to approximately 26 ± 8 mM, demonstrating that under in vivo conditions KCC2 produces low [Cl−]i in adult mouse neurons. In summary, neurons of the brain of awake adult mice exhibit a low and evenly distributed [Cl−]i in the proximal somato-dendritic compartment that is independent of anesthesia and requires KCC2 expression for its maintenance.

Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe.

Science.gov (United States)

Li, Cuixia; Zuo, Jing; Zhang, Li; Chang, Yulei; Zhang, Youlin; Tu, Langping; Liu, Xiaomin; Xue, Bin; Li, Qiqing; Zhao, Huiying; Zhang, Hong; Kong, Xianggui

2016-12-09

Accurate quantitation of intracellular pH (pH i ) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pH i sensing. Till now, biological auto-fluorescence background upon UV-Vis excitation and severe photo-bleaching of dyes are the two main factors impeding the accurate quantitative detection of pH i . Herein, we have developed a self-ratiometric luminescence nanoprobe based on förster resonant energy transfer (FRET) for probing pH i , in which pH-sensitive fluorescein isothiocyanate (FITC) and upconversion nanoparticles (UCNPs) were served as energy acceptor and donor, respectively. Under 980 nm excitation, upconversion emission bands at 475 nm and 645 nm of NaYF 4 :Yb 3+ , Tm 3+ UCNPs were used as pH i response and self-ratiometric reference signal, respectively. This direct quantitative sensing approach has circumvented the traditional software-based subsequent processing of images which may lead to relatively large uncertainty of the results. Due to efficient FRET and fluorescence background free, a highly-sensitive and accurate sensing has been achieved, featured by 3.56 per unit change in pH i value 3.0-7.0 with deviation less than 0.43. This approach shall facilitate the researches in pH i related areas and development of the intracellular drug delivery systems.

Sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured rat caput epididymal epithelium.

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Wu-Lin Zuo

Full Text Available The epithelium lining the epididymis provides an optimal acidic fluid microenvironment in the epididymal tract that enable spermatozoa to complete the maturation process. The present study aims to investigate the functional role of Na(+/HCO(3(- cotransporter in the pH regulation in rat epididymis.Immunofluorescence staining of pan cytokeratin in the primary culture of rat caput epididymal epithelium showed that the system was a suitable model for investigating the function of epididymal epithelium. Intracellular and apical pH were measured using the fluorescent pH sensitive probe carboxy-seminaphthorhodafluor-4F acetoxymethyl ester (SNARF-4F and sparklet pH electrode respectively to explore the functional role of rat epididymal epithelium. In the HEPES buffered Krebs-Henseleit (KH solution, the intracellular pH (pHi recovery from NH(4Cl induced acidification in the cultured caput epididymal epithelium was completely inhibited by amiloride, the inhibitor of Na(+/H(+ exchanger (NHE. Immediately changing of the KH solution from HEPES buffered to HCO(3(- buffered would cause another pHi recovery. The pHi recovery in HCO(3(- buffered KH solution was inhibited by 4, 4diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, the inhibitor of HCO(3(- transporter or by removal of extracellular Na(+. The extracellular pH measurement showed that the apical pH would increase when adding DIDS to the apical side of epididymal epithelial monolayer, however adding DIDS to the basolateral side had no effect on apical pH.The present study shows that sodium coupled bicarbonate influx regulates intracellular and apical pH in cultured caput epididymal epithelium.

In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system.

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Martinière, Alexandre; Bassil, Elias; Jublanc, Elodie; Alcon, Carine; Reguera, Maria; Sentenac, Hervé; Blumwald, Eduardo; Paris, Nadine

2013-10-01

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

Intracellular pH regulation in hepatocytes isolated from three teleost species.

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Furimsky, M; Moon, T W; Perry, S F

1999-09-01

The mechanisms of intracellular pH (pH(i)) regulation were studied in hepatocytes isolated from three species of teleost: rainbow trout (Oncorhynchus mykiss), black bullhead (Ameiurus melas) and American eel (Anguilla rostrata). Intracellular pH was monitored over time using the pH-sensitive fluorescent dye BCECF in response to acid loading under control conditions and in different experimental media containing either low Na(+) or Cl(-) concentrations, the Na(+)-H(+) exchanger blocker amiloride or the blocker of the V-type H(+)-ATPase, bafilomycin A(1). In trout and bullhead hepatocytes, recovery to an intracellular acid load occurred principally by way of a Na(+)-dependent amiloride-sensitive Na(+)-H(+) exchanger. In eel hepatocytes, the Na(+)-H(+) exchanger did not contribute to recovery to an acid load though evidence suggests that it is present on the cell membrane and participates in the maintenance of steady-state pH(i). The V-type H(+)-ATPase did not participate in recovery to an acid load in any species. A Cl(-)-HCO(3)(-) exchanger may play a role in recovery to an acid load in eel hepatocytes by switching off and retaining base that would normally be tonically extruded. Thus, it is clear that hepatocytes isolated from the three species are capable of regulating pH(i), principally by way of a Na(+)-H(+) exchanger and a Cl(-)-HCO(3)(-) exchanger, but do not exploit identical mechanisms for pH(i) recovery. J. Exp. Zool. 284:361-367, 1999. Copyright 1999 Wiley-Liss, Inc.

[Alpha but not beta-adrenergic stimulation has a positive inotropic effect associated with alkalinization of intracellular pH].

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Gambassi, G; Lakatta, E G; Capogrossi, M C

1991-01-01

There is increasing evidence that alpha-adrenoceptors also exist in the myocardium and that an increase in force of contraction may be produced by stimulation of these sites. This positive inotropism seems to be dependent either on an increased amount of Ca++ released into the cytosol with each action potential or on increased myofilament responsiveness. In contrast, beta-adrenergic stimulation reduces the sensitivity of the contractile proteins and the positive inotropic effect is due to the activation of L-type calcium channels on the sarcolemma. We used single, isolated, enzymatically dissociated, adult rat ventricular myocytes. Cells were loaded either with the ester derivative of the Ca++ probe Indo-1 or with the intracellular pH probe Snarf-1 and at the same time we measured the contractile parameters and monitored the fluorescence as an index of intracellular calcium concentration or pH value. The single cells (bicarbonate buffer continuously gassed with O2 95%, CO2 5%, Ca++ 1.5 mM, field stimulation 0.5 Hz) were exposed to phenylephrine (50 microM) and nadolol (1 microM). Alpha-adrenergic stimulation increased twitch amplitude (delta ES = 1.93 +/- 0.77, n = 8; p less than 0.05) and showed only a slight increase in Ca++ transient. On the other end, the positive inotropic effect (delta ES = 2.84 +/- 0.86, n = 4; p less than 0.02) obtained with beta-adrenergic stimulation (isoproterenol 50 nM, bicarbonate buffer, Ca++ 0.5 mM, field stimulation 0.2 Hz) was always associated with a large increase in intracellular Ca++ concentration. Isoproterenol did not change intracellular pH (delta pH = 0.006 +/- 0.006, n = 4; NS) while phenylephrine increased it significantly (delta pH = 0.055 +/- 0.011, n = 8; p less than 0.002). Moreover, there was a statistically significant correlation between delta ES and delta pH (R2 = 0.532; p less than 0.05) when phenylephrine was present. This alkalinization as well as the increased contractility was antagonized by treatment with

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium.

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Walker, Nancy M; Liu, Jinghua; Stein, Sydney R; Stefanski, Casey D; Strubberg, Ashlee M; Clarke, Lane L

2016-01-15

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl(-) and HCO3 (-) efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3 (-))-loading proteins and upregulation of the basolateral membrane HCO3 (-)-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl(-)/HCO3 (-) exchange with maximized gradients, it also had increased intracellular Cl(-) concentration relative to wild-type. Pharmacological reduction of intracellular Cl(-) concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl(-) and HCO3 (-) efflux, which impairs pHi regulation by Ae2. Retention of Cl(-) and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. Copyright © 2016 the American Physiological Society.

Cellular chloride and bicarbonate retention alters intracellular pH regulation in Cftr KO crypt epithelium

Science.gov (United States)

Walker, Nancy M.; Liu, Jinghua; Stein, Sydney R.; Stefanski, Casey D.; Strubberg, Ashlee M.

2015-01-01

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), an anion channel providing a major pathway for Cl− and HCO3− efflux across the apical membrane of the epithelium. In the intestine, CF manifests as obstructive syndromes, dysbiosis, inflammation, and an increased risk for gastrointestinal cancer. Cftr knockout (KO) mice recapitulate CF intestinal disease, including intestinal hyperproliferation. Previous studies using Cftr KO intestinal organoids (enteroids) indicate that crypt epithelium maintains an alkaline intracellular pH (pHi). We hypothesized that Cftr has a cell-autonomous role in downregulating pHi that is incompletely compensated by acid-base regulation in its absence. Here, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorimetry of enteroids showed that Cftr KO crypt epithelium sustains an alkaline pHi and resistance to cell acidification relative to wild-type. Quantitative real-time PCR revealed that Cftr KO enteroids exhibit downregulated transcription of base (HCO3−)-loading proteins and upregulation of the basolateral membrane HCO3−-unloader anion exchanger 2 (Ae2). Although Cftr KO crypt epithelium had increased Ae2 expression and Ae2-mediated Cl−/HCO3− exchange with maximized gradients, it also had increased intracellular Cl− concentration relative to wild-type. Pharmacological reduction of intracellular Cl− concentration in Cftr KO crypt epithelium normalized pHi, which was largely Ae2-dependent. We conclude that Cftr KO crypt epithelium maintains an alkaline pHi as a consequence of losing both Cl− and HCO3− efflux, which impairs pHi regulation by Ae2. Retention of Cl− and an alkaline pHi in crypt epithelium may alter several cellular processes in the proliferative compartment of Cftr KO intestine. PMID:26542396

Stream pH as an abiotic gradient influencing distributions of trout in Pennsylvania streams

Science.gov (United States)

Kocovsky, P.M.; Carline, R.F.

2005-01-01

Elevation and stream slope are abiotic gradients that limit upstream distributions of brook trout Salvelinus fontinalis and brown trout Salmo trutta in streams. We sought to determine whether another abiotic gradient, base-flow pH, may also affect distributions of these two species in eastern North America streams. We used historical data from the Pennsylvania Fish and Boat Commission's fisheries management database to explore the effects of reach elevation, slope, and base-flow pH on distributional limits to brook trout and brown trout in Pennsylvania streams in the Appalachian Plateaus and Ridge and Valley physiographic provinces. Discriminant function analysis (DFA) was used to calculate a canonical axis that separated allopatric brook trout populations from allopatric brown trout populations and allowed us to assess which of the three independent variables were important gradients along which communities graded from allopatric brook trout to allopatric brown trout. Canonical structure coefficients from DFA indicated that in both physiographic provinces, stream base-flow pH and slope were important factors in distributional limits; elevation was also an important factor in the Ridge and Valley Province but not the Appalachian Plateaus Province. Graphs of each variable against the proportion of brook trout in a community also identified apparent zones of allopatry for both species on the basis of pH and stream slope. We hypothesize that pH-mediated interspecific competition that favors brook trout in competition with brown trout at lower pH is the most plausible mechanism for segregation of these two species along pH gradients. Our discovery that trout distributions in Pennsylvania are related to stream base-flow pH has important implications for brook trout conservation in acidified regions. Carefully designed laboratory and field studies will be required to test our hypothesis and elucidate the mechanisms responsible for the partitioning of brook trout and

Modelling the extra and intracellular uptake and discharge of heavy metals in Fontinalis antipyretica transplanted along a heavy metal and pH contamination gradient

International Nuclear Information System (INIS)

Fernandez, J.A.; Vazquez, M.D.; Lopez, J.; Carballeira, A.

2006-01-01

Samples of the aquatic bryophyte Fontinalis antipyretica Hedw. were transplanted to different sites with the aim of characterizing the kinetics of the uptake and discharge of heavy metals in the extra and intracellular compartments. The accumulation of metals in extracellular compartments, characterized by an initial rapid accumulation, then a gradual slowing down over time, fitted perfectly to a Michaelis-Menten model. The discharge of metals from the same compartment followed an inverse linear model or an inverse Michaelis-Menten model, depending on the metal. In intracellular sites both uptake and discharge occurred more slowly and progressively, following a linear model. We also observed that the acidity of the environment greatly affected metal accumulation in extracellular sites, even when the metals were present at relatively high concentrations, whereas the uptake of metals within cells was much less affected by pH. - The kinetics of uptake and discharge of heavy metals, in different cellular locations, were studied in transplanted aquatic mosses

Spontaneous formation of small unilamellar vesicles by pH jump: A pH gradient across the bilayer membrane as the driving force

International Nuclear Information System (INIS)

Hauser, H.; Mantsch, H.H.; Casal, H.L.

1990-01-01

31 P NMR and infrared spectroscopic methods have been used to study the formation of small unilamellar vesicles by the pH-jump method. It is shown that increasing the pH of different lamellar phospholipid dispersions (phosphatidic acids and phosphatidylserines) induces a pH gradient. This pH gradient is estimated to be 4 ± 1 pH units, and its direction is such that the inner monolayer of the vesicles is at lower pH. There is spectroscopic evidence for tighter packing of the lipid hydrocarbon chains in the inner monolayer, probably due to the constraints imposed by the high curvature of the small vesicles formed. These results are discussed in terms of the driving force of the spontaneous vesiculation

Ratiometric Imaging of Extracellular pH in Dental Biofilms

DEFF Research Database (Denmark)

Schlafer, Sebastian; Dige, Irene

2016-01-01

The pH in bacterial biofilms on teeth is of central importance for dental caries, a disease with a high worldwide prevalence. Nutrients and metabolites are not distributed evenly in dental biofilms. A complex interplay of sorption to and reaction with organic matter in the biofilm reduces...... the diffusion paths of solutes and creates steep gradients of reactive molecules, including organic acids, across the biofilm. Quantitative fluorescent microscopic methods, such as fluorescence life time imaging or pH ratiometry, can be employed to visualize pH in different microenvironments of dental biofilms...... allows monitoring both vertical and horizontal pH gradients in real-time without mechanically disturbing the biofilm. However, care must be taken to differentiate accurately between extra- and intracellular compartments of the biofilm. Here, the ratiometric dye, seminaphthorhodafluor-4F 5-(and-6...

Participation of intracellular and extracellular pH changes in photosynthetic response development induced by variation potential in pumpkin seedlings.

Science.gov (United States)

Sherstneva, O N; Vodeneev, V A; Katicheva, L A; Surova, L M; Sukhov, V S

2015-06-01

Electrical signals presented in plants by action potential and by variation potential (VP) can induce a reversible inactivation of photosynthesis. Changes in the intracellular and extracellular pH during VP generation are a potential mechanism of photosynthetic response induction; however, this hypothesis requires additional experimental investigation. The purpose of the present work was to analyze the influence of pH changes on induction of the photosynthetic response in pumpkin. It was shown that a burning of the cotyledon induced VP propagation into true leaves of pumpkin seedlings inducing a decrease in the photosynthetic CO2 assimilation and an increase in non-photochemical quenching of fluorescence, whereas respiration was activated insignificantly. The photosynthetic response magnitude depended linearly on the VP amplitude. The intracellular and extracellular concentrations of protons were analyzed using pH-sensitive fluorescent probes, and the VP generation was shown to be accompanied by apoplast alkalization (0.4 pH unit) and cytoplasm acidification (0.3 pH unit). The influence of changes in the incubation medium pH on the non-photochemical quenching of fluorescence of isolated chloroplasts was also investigated. It was found that acidification of the medium stimulated the non-photochemical quenching, and the magnitude of this increase depended on the decrease in pH. Our results confirm the contribution of changes in intracellular and extracellular pH to induction of the photosynthetic response caused by VP. Possible mechanisms of the influence of pH changes on photosynthesis are discussed.

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells

NARCIS (Netherlands)

Pandey, R.; Vischer, N.O.E.; Smelt, J.P.P.M.; van Beilen, J.W.A.; Ter Beek, A.; De Vos, W.H.; Brul, S.; Manders, E.M.M.

2016-01-01

Intracellular pH (pHi) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pHi homeostasis. Unfortunately, accurate pHi quantification with existing methods is suboptimal, since measurements are averages across populations of cells,

Accurate Quantitative Sensing of Intracellular pH based on Self-ratiometric Upconversion Luminescent Nanoprobe

NARCIS (Netherlands)

Li, C.; Zuo, J.; Zhang, L.; Chang, Y.; Zhang, Y.; Tu, L.; Liu, X.; Xue, B.; Li, Q.; Zhao, H.; Zhang, H.; Kong, X.

2016-01-01

Accurate quantitation of intracellular pH (pHi) is of great importance in revealing the cellular activities and early warning of diseases. A series of fluorescence-based nano-bioprobes composed of different nanoparticles or/and dye pairs have already been developed for pHi sensing. Till now,

Live-cell Microscopy and Fluorescence-based Measurement of Luminal pH in Intracellular Organelles

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Li Ma

2017-08-01

Full Text Available Luminal pH is an important functional feature of intracellular organelles. Acidification of the lumen of organelles such as endosomes, lysosomes, and the Golgi apparatus plays a critical role in fundamental cellular processes. As such, measurement of the luminal pH of these organelles has relevance to both basic research and translational research. At the same time, accurate measurement of intraorganellar pH in living cells can be challenging and may be a limiting hurdle for research in some areas. Here, we describe three powerful methods to measure rigorously the luminal pH of different intracellular organelles, focusing on endosomes, lysosomes, and the Golgi apparatus. The described methods are based on live imaging of pH-sensitive fluorescent probes and include: (1 A protocol based on quantitative, ratiometric measurement of endocytosis of pH-sensitive and pH-insensitive fluorescent conjugates of transferrin; (2 A protocol for the use of proteins tagged with a ratiometric variant of the pH-sensitive intrinsically fluorescent protein pHluorin; and (3 A protocol using the fluorescent dye LysoSensor™. We describe necessary reagents, key procedures, and methods and equipment for data acquisition and analysis. Examples of implementation of the protocols are provided for cultured cells derived from a cancer cell line and for primary cultures of mouse hippocampal neurons. In addition, we present strengths and weaknesses of the different described intraorganellar pH measurement methods. These protocols are likely to be of benefit to many researchers, from basic scientists to those conducting translational research with a focus on diseases in patient-derived cells.

Capturing intracellular pH dynamics by coupling its molecular mechanisms within a fully tractable mathematical model.

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Yann Bouret

Full Text Available We describe the construction of a fully tractable mathematical model for intracellular pH. This work is based on coupling the kinetic equations depicting the molecular mechanisms for pumps, transporters and chemical reactions, which determine this parameter in eukaryotic cells. Thus, our system also calculates the membrane potential and the cytosolic ionic composition. Such a model required the development of a novel algebraic method that couples differential equations for slow relaxation processes to steady-state equations for fast chemical reactions. Compared to classical heuristic approaches based on fitted curves and ad hoc constants, this yields significant improvements. This model is mathematically self-consistent and allows for the first time to establish analytical solutions for steady-state pH and a reduced differential equation for pH regulation. Because of its modular structure, it can integrate any additional mechanism that will directly or indirectly affect pH. In addition, it provides mathematical clarifications for widely observed biological phenomena such as overshooting in regulatory loops. Finally, instead of including a limited set of experimental results to fit our model, we show examples of numerical calculations that are extremely consistent with the wide body of intracellular pH experimental measurements gathered by different groups in many different cellular systems.

The impact of pH inhomogeneities on CHO cell physiology and fed-batch process performance - two-compartment scale-down modelling and intracellular pH excursion.

Science.gov (United States)

Brunner, Matthias; Braun, Philipp; Doppler, Philipp; Posch, Christoph; Behrens, Dirk; Herwig, Christoph; Fricke, Jens

2017-07-01

Due to high mixing times and base addition from top of the vessel, pH inhomogeneities are most likely to occur during large-scale mammalian processes. The goal of this study was to set-up a scale-down model of a 10-12 m 3 stirred tank bioreactor and to investigate the effect of pH perturbations on CHO cell physiology and process performance. Short-term changes in extracellular pH are hypothesized to affect intracellular pH and thus cell physiology. Therefore, batch fermentations, including pH shifts to 9.0 and 7.8, in regular one-compartment systems are conducted. The short-term adaption of the cells intracellular pH are showed an immediate increase due to elevated extracellular pH. With this basis of fundamental knowledge, a two-compartment system is established which is capable of simulating defined pH inhomogeneities. In contrast to state-of-the-art literature, the scale-down model is included parameters (e.g. volume of the inhomogeneous zone) as they might occur during large-scale processes. pH inhomogeneity studies in the two-compartment system are performed with simulation of temporary pH zones of pH 9.0. The specific growth rate especially during the exponential growth phase is strongly affected resulting in a decreased maximum viable cell density and final product titer. The gathered results indicate that even short-term exposure of cells to elevated pH values during large-scale processes can affect cell physiology and overall process performance. In particular, it could be shown for the first time that pH perturbations, which might occur during the early process phase, have to be considered in scale-down models of mammalian processes. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of the intracellular pH of intact erythrocytes by 1H NMR spectroscopy

International Nuclear Information System (INIS)

Rabenstein, D.L.; Isab, A.A.

1982-01-01

A method is described for determining the intracellular pH of intact erythrocytes by 1 H NMR. The determination is based on the pH dependence of the chemical shifts of resonances for carbon-bounded protons of an indicator molecule (imidazole) in intact cells. The imidazole is introduced into the erythrocytes by incubation in an isotonic saline solution of the indicator. The pH dependence of the chemical shifts of the imidazole resonances is calibrated from 1 H NMR spectra of the imidazole-containing red cell lysates whose pH is varied by the addition of acid or base and measured directly with a pH electrode. To reduce in intensity or eliminate the much more intense envelope of resonances from the hemoglobin, the 1 H NMR measurements are made by either the spin-echo Fourier transform technique or by the transfer-or-saturation by cross-relaxation method

In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth

NARCIS (Netherlands)

Orij, R.; Postmus, J.; ter Beek, A.; Brul, S.; Smits, G.J.

2009-01-01

The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartment-specific measurements of intracellular pH (pHi) dynamics in living cells are therefore crucial to the

The Effect of Curcumin on Intracellular pH (pHi), Membrane Hyperpolarization and Sperm Motility.

Science.gov (United States)

Naz, Rajesh K

2014-04-01

Curcumin has shown to affect sperm motility and function in vitro and fertility in vivo. The molecular mechanism(s) by which curcumin affects sperm motility has not been delineated. Since modulation of intracellular pH (pHi) and plasma membrane polarization is involved in sperm motility, the present study was conducted to investigate the effect of curcumin on these sperm (human and murine) parameters. The effect of curcumin on sperm forward motility was examined by counting percentages of forward moving sperm. The effect of curcumin on intracellular pH (pHi) was measured by the fluorescent pH indicator 2,7-bicarboxyethyl-5,6-carboxyfluorescein-acetoxymethyl ester (BCECF-AM). The effect of curcumin on plasma membrane polarization was examined using the fluorescence sensitive dye bis (1,3-dibarbituric acid)-trimethine oxanol [DiBAC4(3)]. Curcumin caused a concentration-dependent (ppHi) in both human and mouse sperm. Curcumin induced significant (ppHi and membrane polarization that affect sperm forward motility. These exciting findings will have application in deciphering the signal transduction pathway involved in sperm motility and function and in development of a novel non-steroidal contraceptive for infertility.

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells.

Science.gov (United States)

Pandey, Rachna; Vischer, Norbert O E; Smelt, Jan P P M; van Beilen, Johan W A; Ter Beek, Alexander; De Vos, Winnok H; Brul, Stanley; Manders, Erik M M

2016-11-01

Intracellular pH (pH i ) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pH i homeostasis. Unfortunately, accurate pH i quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity. Yet, physiological heterogeneity in isogenic populations is well known to be responsible for differences in growth and division kinetics of cells in response to external stressors. To assess in this context the behavior of intracellular acidity, we have developed a robust method to quantify pH i at single-cell levels in Bacillus subtilis Bacilli spoil food, cause disease, and are well known for their ability to form highly stress-resistant spores. Using an improved version of the genetically encoded ratiometric pHluorin (IpHluorin), we have quantified pH i in individual B. subtilis cells, cultured at an external pH of 6.4, in the absence or presence of weak acid stresses. In the presence of 3 mM potassium sorbate, a decrease in pH i and an increase in the generation time of growing cells were observed. Similar effects were observed when cells were stressed with 25 mM potassium acetate. Time-resolved analysis of individual bacteria in growing colonies shows that after a transient pH decrease, long-term pH evolution is highly cell dependent. The heterogeneity at the single-cell level shows the existence of subpopulations that might be more resistant and contribute to population survival. Our approach contributes to an understanding of pH i regulation in individual bacteria and may help scrutinizing effects of existing and novel food preservation strategies. This study shows how the physiological response to commonly used weak organic acid food preservatives, such as sorbic and acetic acids, can be measured at the single-cell level. These data are key to coupling often-observed single-cell heterogeneous growth

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations

DEFF Research Database (Denmark)

Christensen, Henriette L; Păunescu, Teodor G; Matchkov, Vladimir

2017-01-01

fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na(+)-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7...

Nestedness in Arbuscular Mycorrhizal Fungal Communities along Soil pH Gradients in Early Primary Succession: Acid-Tolerant Fungi Are pH Generalists.

Science.gov (United States)

Kawahara, Ai; An, Gi-Hong; Miyakawa, Sachie; Sonoda, Jun; Ezawa, Tatsuhiro

2016-01-01

Soil acidity is a major constraint on plant productivity. Arbuscular mycorrhizal (AM) fungi support plant colonization in acidic soil, but soil acidity also constrains fungal growth and diversity. Fungi in extreme environments generally evolve towards specialists, suggesting that AM fungi in acidic soil are acidic-soil specialists. In our previous surveys, however, some AM fungi detected in strongly acidic soils could also be detected in a soil with moderate pH, which raised a hypothesis that the fungi in acidic soils are pH generalists. To test the hypothesis, we conducted a pH-manipulation experiment and also analyzed AM fungal distribution along a pH gradient in the field using a synthesized dataset of the previous and recent surveys. Rhizosphere soils of the generalist plant Miscanthus sinensis were collected both from a neutral soil and an acidic soil, and M. sinensis seedlings were grown at three different pH. For the analysis of field communities, rhizosphere soils of M. sinensis were collected from six field sites across Japan, which covered a soil pH range of 3.0-7.4, and subjected to soil trap culture. AM fungal community compositions were determined based on LSU rDNA sequences. In the pH-manipulation experiment the acidification of medium had a significant impact on the compositions of the community from the neutral soil, but the neutralization of the medium had no effect on those of the community from the acidic soil. Furthermore, the communities in lower -pH soils were subsets of (nested in) those in higher-pH soils. In the field communities a significant nestedness pattern was observed along the pH gradient. These observations suggest that the fungi in strongly acidic soils are pH generalists that occur not only in acidic soil but also in wide ranges of soil pH. Nestedness in AM fungal community along pH gradients may have important implications for plant community resilience and early primary succession after disturbance in acidic soils.

Nestedness in Arbuscular Mycorrhizal Fungal Communities along Soil pH Gradients in Early Primary Succession: Acid-Tolerant Fungi Are pH Generalists

Science.gov (United States)

Kawahara, Ai; An, Gi-Hong; Miyakawa, Sachie; Sonoda, Jun

2016-01-01

Soil acidity is a major constraint on plant productivity. Arbuscular mycorrhizal (AM) fungi support plant colonization in acidic soil, but soil acidity also constrains fungal growth and diversity. Fungi in extreme environments generally evolve towards specialists, suggesting that AM fungi in acidic soil are acidic-soil specialists. In our previous surveys, however, some AM fungi detected in strongly acidic soils could also be detected in a soil with moderate pH, which raised a hypothesis that the fungi in acidic soils are pH generalists. To test the hypothesis, we conducted a pH-manipulation experiment and also analyzed AM fungal distribution along a pH gradient in the field using a synthesized dataset of the previous and recent surveys. Rhizosphere soils of the generalist plant Miscanthus sinensis were collected both from a neutral soil and an acidic soil, and M. sinensis seedlings were grown at three different pH. For the analysis of field communities, rhizosphere soils of M. sinensis were collected from six field sites across Japan, which covered a soil pH range of 3.0–7.4, and subjected to soil trap culture. AM fungal community compositions were determined based on LSU rDNA sequences. In the pH-manipulation experiment the acidification of medium had a significant impact on the compositions of the community from the neutral soil, but the neutralization of the medium had no effect on those of the community from the acidic soil. Furthermore, the communities in lower -pH soils were subsets of (nested in) those in higher-pH soils. In the field communities a significant nestedness pattern was observed along the pH gradient. These observations suggest that the fungi in strongly acidic soils are pH generalists that occur not only in acidic soil but also in wide ranges of soil pH. Nestedness in AM fungal community along pH gradients may have important implications for plant community resilience and early primary succession after disturbance in acidic soils. PMID

Effect of a pH Gradient on the Protonation States of Cytochrome c Oxidase: A Continuum Electrostatics Study.

Science.gov (United States)

Magalhães, Pedro R; Oliveira, A Sofia F; Campos, Sara R R; Soares, Cláudio M; Baptista, António M

2017-02-27

Cytochrome c oxidase (CcO) couples the reduction of dioxygen to water with transmembrane proton pumping, which leads to the generation of an electrochemical gradient. In this study we analyze how one of the components of the electrochemical gradient, the difference in pH across the membrane, or ΔpH, influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study of CcO in the presence of a pH gradient. The inclusion of a transmembrane pH gradient allows for the identification of residues whose titration behavior depends on the pH on both sides of the membrane. Among the several residues with unusual titration profiles, three are well-known key residues in the proton transfer process of CcO: E286 I , Y288 I , and K362 I . All three residues have been previously identified as being critical for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these residues may be part of a regulatory mechanism to stem the proton flow.

Monitoring Intracellular pH change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells

OpenAIRE

Zhang, Yunfei; Robertson, J. Brian; Xie, Qiguang; Johnson, Carl Hirschie

2016-01-01

“pHlash” is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characteri...

Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

Science.gov (United States)

Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

2012-04-15

Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

Modulation of Acid-sensing Ion Channel 1a by Intracellular pH and Its Role in Ischemic Stroke.

Science.gov (United States)

Li, Ming-Hua; Leng, Tian-Dong; Feng, Xue-Chao; Yang, Tao; Simon, Roger P; Xiong, Zhi-Gang

2016-08-26

An important contributor to brain ischemia is known to be extracellular acidosis, which activates acid-sensing ion channels (ASICs), a family of proton-gated sodium channels. Lines of evidence suggest that targeting ASICs may lead to novel therapeutic strategies for stroke. Investigations of the role of ASICs in ischemic brain injury have naturally focused on the role of extracellular pH in ASIC activation. By contrast, intracellular pH (pHi) has received little attention. This is a significant gap in our understanding because the ASIC response to extracellular pH is modulated by pHi, and activation of ASICs by extracellular protons is paradoxically enhanced by intracellular alkalosis. Our previous studies show that acidosis-induced cell injury in in vitro models is attenuated by intracellular acidification. However, whether pHi affects ischemic brain injury in vivo is completely unknown. Furthermore, whereas ASICs in native neurons are composed of different subunits characterized by distinct electrophysiological/pharmacological properties, the subunit-dependent modulation of ASIC activity by pHi has not been investigated. Using a combination of in vitro and in vivo ischemic brain injury models, electrophysiological, biochemical, and molecular biological approaches, we show that the intracellular alkalizing agent quinine potentiates, whereas the intracellular acidifying agent propionate inhibits, oxygen-glucose deprivation-induced cell injury in vitro and brain ischemia-induced infarct volume in vivo Moreover, we find that the potentiation of ASICs by quinine depends on the presence of the ASIC1a, ASIC2a subunits, but not ASIC1b, ASIC3 subunits. Furthermore, we have determined the amino acids in ASIC1a that are involved in the modulation of ASICs by pHi. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Radiation-induced apoptosis of stem/progenitor cells in human umbilical cord blood is associated with alterations in reactive oxygen and intracellular pH

International Nuclear Information System (INIS)

Hayashi, Tomonori; Hayashi, Ikue; Shinohara, Tomoko; Morishita, Yukari; Nagamura, Hiroko; Kusunoki, Yoichiro; Kyoizumi, Seishi; Seyama, Toshio; Nakachi, Kei

2004-01-01

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34 + /CD38 - , CD34 + /CD38 + and CD34 - /CD38 + cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34 + /CD38 - cell population, where the level of O2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34 + /CD38 - cell population, and this intracellular pH decreased as early as 4h post-irradiation, virtually simultaneous with the significant elevation of O2- generation. These results suggest that the CD34 + /CD38 - stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O2-, compare to more differentiated CD34 + /CD38 + and CD34 - /CD38 + cells and that its intracellular pH declines at an early phase in the apoptosis process

Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients

NARCIS (Netherlands)

Bischoff, Rainer; Roecklin, D.; Roitsch, C.

1992-01-01

Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase

Monitoring Intracellular pH Change with a Genetically Encoded and Ratiometric Luminescence Sensor in Yeast and Mammalian Cells.

Science.gov (United States)

Zhang, Yunfei; Robertson, J Brian; Xie, Qiguang; Johnson, Carl Hirschie

2016-01-01

"pHlash" is a novel bioluminescence-based pH sensor for measuring intracellular pH, which is developed based on Bioluminescence Resonance Energy Transfer (BRET). pHlash is a fusion protein between a mutant of Renilla luciferase (RLuc) and a Venus fluorophore. The spectral emission of purified pHlash protein exhibits pH dependence in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification. In this chapter, we describe an in vitro characterization of pHlash, and also in vivo assays including in yeast cells and in HeLa cells using pHlash as a cytoplasmic pH indicator.

Archaeal abundance across a pH gradient in an arable soil and its relationship to bacterial and fungal growth rates.

Science.gov (United States)

Bengtson, Per; Sterngren, Anna E; Rousk, Johannes

2012-08-01

Soil pH is one of the most influential factors for the composition of bacterial and fungal communities, but the influence of soil pH on the distribution and composition of soil archaeal communities has yet to be systematically addressed. The primary aim of this study was to determine how total archaeal abundance (quantitative PCR [qPCR]-based estimates of 16S rRNA gene copy numbers) is related to soil pH across a pH gradient (pH 4.0 to 8.3). Secondarily, we wanted to assess how archaeal abundance related to bacterial and fungal growth rates across the same pH gradient. We identified two distinct and opposite effects of pH on the archaeal abundance. In the lowest pH range (pH 4.0 to 4.7), the abundance of archaea did not seem to correspond to pH. Above this pH range, there was a sharp, almost 4-fold decrease in archaeal abundance, reaching a minimum at pH 5.1 to 5.2. The low abundance of archaeal 16S rRNA gene copy numbers at this pH range then sharply increased almost 150-fold with pH, resulting in an increase in the ratio between archaeal and bacterial copy numbers from a minimum of 0.002 to more than 0.07 at pH 8. The nonuniform archaeal response to pH could reflect variation in the archaeal community composition along the gradient, with some archaea adapted to acidic conditions and others to neutral to slightly alkaline conditions. This suggestion is reinforced by observations of contrasting outcomes of the (competitive) interactions between archaea, bacteria, and fungi toward the lower and higher ends of the examined pH gradient.

Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites

International Nuclear Information System (INIS)

Louisse, Jochem; Bai Yanqing; Verwei, Miriam; Sandt, Johannes J.M. van de; Blaauboer, Bas J.; Rietjens, Ivonne M.C.M.

2010-01-01

Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH i ) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH i in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH i of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na + /H + -antiporter, corroborating an important role of the pH i in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH i may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

Radiation-induced apoptosis of stem/progenitor cells in human umbilical cord blood is associated with alterations in reactive oxygen and intracellular pH

Energy Technology Data Exchange (ETDEWEB)

Hayashi, Tomonori [Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hijyama Park, Minami Ward, Hiroshima (Japan)]. E-mail: tomo@rerf.or.jp; Hayashi, Ikue [Central Research Laboratory, Hiroshima University Faculty of Dentistry, Hiroshima (Japan); Shinohara, Tomoko [Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hijyama Park, Minami Ward, Hiroshima (Japan); Morishita, Yukari [Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hijyama Park, Minami Ward, Hiroshima (Japan); Nagamura, Hiroko [Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hijyama Park, Minami Ward, Hiroshima (Japan); Kusunoki, Yoichiro [Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hijyama Park, Minami Ward, Hiroshima (Japan); Kyoizumi, Seishi [Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hijyama Park, Minami Ward, Hiroshima (Japan); Seyama, Toshio [Yasuda Women' s University, Hiroshima (Japan); Nakachi, Kei [Department of Radiobiology/Molecular Epidemiology, Radiation Effects Research Foundation, Hijyama Park, Minami Ward, Hiroshima (Japan)

2004-11-22

To investigate the sensitivity of human hematopoietic stem cell populations to radiation and its relevance to intracellular events, specifically alteration in cellular energy production systems, we examined the frequency of apoptotic cells, generation of superoxide anions (O2-), and changes in cytosol pH in umbilical cord blood (UCB) CD34{sup +}/CD38{sup -}, CD34{sup +}/CD38{sup +} and CD34{sup -}/CD38{sup +} cells before and after 5Gy of X-irradiation. Human UCB mononucleated cells were used in this study. After X-irradiation and staining subgroups of the cells with fluorescence (FITC, PE, or CY)-labeled anti-CD34 and anti-CD38 antibodies, analyses were performed by FACScan using as stains 7-amino-actinomycin D (7-AAD) for the detection of apoptosis, and hydroethidine (HE) for the measurement of O2- generation in the cells. For intracellular pH, image analysis was conducted using confocal laser microscopy after irradiation and staining with carboxy-SNAFR-1. The frequency of apoptotic cells, as determined by cell staining with 7-AAD, was highest in the irradiated CD34{sup +}/CD38{sup -} cell population, where the level of O2- detected by the oxidation of HE was also most highly elevated. Intracellular pH measured with carboxy-SNARF-1-AM by image cytometer appeared to be lowest in the same irradiated CD34{sup +}/CD38{sup -} cell population, and this intracellular pH decreased as early as 4h post-irradiation, virtually simultaneous with the significant elevation of O2- generation. These results suggest that the CD34{sup +}/CD38{sup -} stem cell population is sensitive to radiation-induced apoptosis as well as production of intracellular O2-, compare to more differentiated CD34{sup +}/CD38{sup +} and CD34{sup -}/CD38{sup +} cells and that its intracellular pH declines at an early phase in the apoptosis process.

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases.

Science.gov (United States)

Tsonev, Latchezar I; Hirsh, Allen G

2008-07-25

pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt.

Role of Cl- -HCO3- exchanger AE3 in intracellular pH homeostasis in cultured murine hippocampal neurons, and in crosstalk to adjacent astrocytes.

Science.gov (United States)

Salameh, Ahlam I; Hübner, Christian A; Boron, Walter F

2017-01-01

A polymorphism of human AE3 is associated with idiopathic generalized epilepsy. Knockout of AE3 in mice lowers the threshold for triggering epileptic seizures. The explanations for these effects are elusive. Comparisons of cells from wild-type vs. AE3 -/- mice show that AE3 (present in hippocampal neurons, not astrocytes; mediates HCO 3 - efflux) enhances intracellular pH (pH i ) recovery (decrease) from alkali loads in neurons and, surprisingly, adjacent astrocytes. During metabolic acidosis (MAc), AE3 speeds initial acidification, but limits the extent of pH i decrease in neurons and astrocytes. AE3 speeds re-alkalization after removal of MAc in neurons and astrocytes, and speeds neuronal pH i recovery from an ammonium prepulse-induced acid load. We propose that neuronal AE3 indirectly increases acid extrusion in (a) neurons via Cl - loading, and (b) astrocytes by somehow enhancing NBCe1 (major acid extruder). The latter would enhance depolarization-induced alkalinization of astrocytes, and extracellular acidification, and thereby reduce susceptibility to epileptic seizures. The anion exchanger AE3, expressed in hippocampal (HC) neurons but not astrocytes, contributes to intracellular pH (pH i ) regulation by facilitating the exchange of extracellular Cl - for intracellular HCO 3 - . The human AE3 polymorphism A867D is associated with idiopathic generalized epilepsy. Moreover, AE3 knockout (AE3 -/- ) mice are more susceptible to epileptic seizure. The mechanism of these effects has been unclear because the starting pH i in AE3 -/- and wild-type neurons is indistinguishable. The purpose of the present study was to use AE3 -/- mice to investigate the role of AE3 in pH i homeostasis in HC neurons, co-cultured with astrocytes. We find that the presence of AE3 increases the acidification rate constant during pH i recovery from intracellular alkaline loads imposed by reducing [CO 2 ]. The presence of AE3 also speeds intracellular acidification during the early phase of

Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors

Directory of Open Access Journals (Sweden)

Mojca Benčina

2013-12-01

Full Text Available Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment, or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiology. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments.

Plasmalemmal V-H+-ATPases regulate intracellular pH in human lung microvascular endothelial cells

International Nuclear Information System (INIS)

Rojas, Jose D.; Sennoune, Souad R.; Maiti, Debasish; Martinez, Gloria M.; Bakunts, Karina; Wesson, Donald E.; Martinez-Zaguilan, Raul

2004-01-01

The lung endothelium layer is exposed to continuous CO 2 transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na + /H + exchanger and HCO 3 - -dependent H + -transporting mechanisms regulate intracellular pH (pH cyt ) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H + -ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na + /H + exchanger and HCO 3 - -based H + -transporting mechanisms, to maintain pH cyt homeostasis. Immunocytochemical studies revealed V-H + -ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na + and HCO 3 - that were similar to those observed in the presence of either Na + , or Na + and HCO 3 - . The Na + - and HCO 3 - -independent pH cyt recovery was inhibited by bafilomycin A 1 , a V-H + -ATPase inhibitor. These studies show a Na + - and HCO 3 - -independent pH cyt regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases

A physics-based model for maintenance of the pH gradient in the gastric mucus layer.

Science.gov (United States)

Lewis, Owen L; Keener, James P; Fogelson, Aaron L

2017-12-01

It is generally accepted that the gastric mucus layer provides a protective barrier between the lumen and the mucosa, shielding the mucosa from acid and digestive enzymes and preventing autodigestion of the stomach epithelium. However, the precise mechanisms that contribute to this protective function are still up for debate. In particular, it is not clear what physical processes are responsible for transporting hydrogen protons, secreted within the gastric pits, across the mucus layer to the lumen without acidifying the environment adjacent to the epithelium. One hypothesis is that hydrogen may be bound to the mucin polymers themselves as they are convected away from the mucosal surface and eventually degraded in the stomach lumen. It is also not clear what mechanisms prevent hydrogen from diffusing back toward the mucosal surface, thereby lowering the local pH. In this work we investigate a physics-based model of ion transport within the mucosal layer based on a Nernst-Planck-like equation. Analysis of this model shows that the mechanism of transporting protons bound to the mucus gel is capable of reproducing the trans-mucus pH gradients reported in the literature. Furthermore, when coupled with ion exchange at the epithelial surface, our analysis shows that bicarbonate secretion alone is capable of neutralizing the epithelial pH, even in the face of enormous diffusive gradients of hydrogen. Maintenance of the pH gradient is found to be robust to a wide array of perturbations in both physiological and phenomenological model parameters, suggesting a robust physiological control mechanism. NEW & NOTEWORTHY This work combines modeling techniques based on physical principles, as well as novel numerical simulations to test the plausibility of one hypothesized mechanism for proton transport across the gastric mucus layer. Results show that this mechanism is able to maintain the extreme pH gradient seen in in vivo experiments and suggests a highly robust regulation

Intracellular chemical gradients: morphing principle in bacteria

Directory of Open Access Journals (Sweden)

Endres Robert G

2012-09-01

Full Text Available Abstract Advances in computational biology allow systematic investigations to ascertain whether internal chemical gradients can be maintained in bacteria – an open question at the resolution limit of fluorescence microscopy. While it was previously believed that the small bacterial cell size and fast diffusion in the cytoplasm effectively remove any such gradient, a new computational study published in BMC Biophysics supports the emerging view that gradients can exist. The study arose from the recent observation that phosphorylated CtrA forms a gradient prior to cell division in Caulobacter crescentus, a bacterium known for its complicated cell cycle. Tropini et al. (2012 postulate that such gradients can provide an internal chemical compass, directing protein localization, cell division and cell development. More specifically, they describe biochemical and physical constraints on the formation of such gradients and explore a number of existing bacterial cell morphologies. These chemical gradients may limit in vitro analyses, and may ensure timing control and robustness to fluctuations during critical stages in cell development.

Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis.

Science.gov (United States)

Sergeeva, Tatiana F; Shirmanova, Marina V; Zlobovskaya, Olga A; Gavrina, Alena I; Dudenkova, Varvara V; Lukina, Maria M; Lukyanov, Konstantin A; Zagaynova, Elena V

2017-03-01

A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Application of linear pH gradients for the modeling of ion exchange chromatography: Separation of monoclonal antibody monomer from aggregates.

Science.gov (United States)

Kluters, Simon; Wittkopp, Felix; Jöhnck, Matthias; Frech, Christian

2016-02-01

The mobile phase pH is a key parameter of every ion exchange chromatography process. However, mechanistic insights into the pH influence on the ion exchange chromatography equilibrium are rare. This work describes a mechanistic model capturing salt and pH influence in ion exchange chromatography. The pH dependence of the characteristic protein charge and the equilibrium constant is introduced to the steric mass action model based on a protein net charge model considering the number of amino acids interacting with the stationary phase. This allows the description of the adsorption equilibrium of the chromatographed proteins as a function of pH. The model parameters were determined for a monoclonal antibody monomer, dimer, and a higher aggregated species based on a manageable set of pH gradient experiments. Without further modification of the model parameters the transfer to salt gradient elution at fixed pH is demonstrated. A lumped rate model was used to predict the separation of the monoclonal antibody monomer/aggregate mixture in pH gradient elution and for a pH step elution procedure-also at increased protein loadings up to 48 g/L packed resin. The presented model combines both salt and pH influence and may be useful for the development and deeper understanding of an ion exchange chromatography separation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

pH gradients in the diffusive boundary layer of subarctic macrophytes

KAUST Repository

Hendriks, Iris E.; Duarte, Carlos M.; Marbà , Nú ria; Krause-Jensen, Dorte

2017-01-01

Highly productive macrophytes produce diurnal and seasonal cycles in CO concentrations modulated by metabolic activity, which cause discrepancies between pH in the bulk water and near seaweed blades, especially when entering the diffusion boundary layer (DBL). Calcifying epiphytic organisms living in this environment are therefore exposed to a different pH environment than that of the water column. To evaluate the actual pH environment on blade surfaces, we measured the thickness of the DBL and pH gradients within it for six subarctic macrophytes: Fucus vesiculosus, Ascophyllum nodosum, Ulva lactuca, Zostera marina, Saccharina longicruris, and Agarum clathratum. We measured pH under laboratory conditions at ambient temperatures (2–3 °C) and slow, stable flow over the blade surface at five light intensities (dark, 30, 50, 100 and 200 µmol photons m s). Boundary layer thickness ranged between 511 and 1632 µm, while the maximum difference in pH (∆pH) between the blade surface and the water column ranged between 0.4 ± 0.14 (average ± SE; Zostera) and 1.2 ± 0.13 (average ± SE; Ulva) pH units. These differences in pH are larger than predictions for pH changes in the bulk water by the end of the century. A simple quadratic model best described the relationship between light intensity and maximum ∆pH, pointing at relatively low optimum PAR of between 28 and 139 µmol photons m s to reach maximum ∆pH. Elevated pH at the blade surface may provide chemical “refugia” for calcifying epiphytic organisms, especially during summer at higher latitudes where photoperiods are long.

pH gradients in the diffusive boundary layer of subarctic macrophytes

KAUST Repository

Hendriks, Iris E.

2017-06-20

Highly productive macrophytes produce diurnal and seasonal cycles in CO concentrations modulated by metabolic activity, which cause discrepancies between pH in the bulk water and near seaweed blades, especially when entering the diffusion boundary layer (DBL). Calcifying epiphytic organisms living in this environment are therefore exposed to a different pH environment than that of the water column. To evaluate the actual pH environment on blade surfaces, we measured the thickness of the DBL and pH gradients within it for six subarctic macrophytes: Fucus vesiculosus, Ascophyllum nodosum, Ulva lactuca, Zostera marina, Saccharina longicruris, and Agarum clathratum. We measured pH under laboratory conditions at ambient temperatures (2–3 °C) and slow, stable flow over the blade surface at five light intensities (dark, 30, 50, 100 and 200 µmol photons m s). Boundary layer thickness ranged between 511 and 1632 µm, while the maximum difference in pH (∆pH) between the blade surface and the water column ranged between 0.4 ± 0.14 (average ± SE; Zostera) and 1.2 ± 0.13 (average ± SE; Ulva) pH units. These differences in pH are larger than predictions for pH changes in the bulk water by the end of the century. A simple quadratic model best described the relationship between light intensity and maximum ∆pH, pointing at relatively low optimum PAR of between 28 and 139 µmol photons m s to reach maximum ∆pH. Elevated pH at the blade surface may provide chemical “refugia” for calcifying epiphytic organisms, especially during summer at higher latitudes where photoperiods are long.

Structure-activity relationships of dimethylsphingosine (DMS) derivatives and their effects on intracellular pH and Ca2+ in the U937 monocyte cell line.

Science.gov (United States)

Chang, Young-Ja; Lee, Yun-Kyung; Lee, Eun-Hee; Park, Jeong-Ju; Chung, Sung-Kee; Im, Dong-Soon

2006-08-01

We recently reported that dimethylsphingosine (DMS), a metabolite of sphingolipids, increased intracellular pH and Ca2+ concentration in U937 human monocytes. In the present study, we found that dimethylphytosphingosine (DMPH) induced the above responses more robustly than DMS. However, phytosphingosine, monomethylphytosphingosine or trimethylsphingosine showed little or no activity. Synthetic C3 deoxy analogues of sphingosine did show similar activities, with the C16 analogue more so than C18. The following structure-activity relationships were observed between DMS derivatives and the intracellular pH and Ca2+ concentrations in U937 monocytes; 1) dimethyl modification is important for the DMS-induced increase of intracellular pH and Ca2+, 2) the addition of an OH group on C4 enhances both activities, 3) the deletion of the OH group on C3 has a negligible effect on the activities, and 4) C16 appears to be more effective than C18. We also found that W-7, a calmodulin inhibitor, blocked the DMS-induced pH increase, whereas, KN-62, ML9, and MMPX, specific inhibitors for calmodulin-dependent kinase II, myosin light chain kinase, and Ca(2+)-calmodulin-dependent phosphodiesterase, respectively, did not affect DMS-induced increases of pH in the U937 monocytes.

The influence of low dose irradiation on intracellular pH level, synthesizing activity and ATP level in cultured chinese fibroblasts

International Nuclear Information System (INIS)

Parkhomenko, I.M.; Perishvili, G.V.; Turovetskij, V.B.; Kudryashov, Yu.B.; Rubin, A.B.; Brovko, L.Yu.

1993-01-01

X-irradiation of Chinese fibroblasts with doses of 0.05-0.15 Gy was shown to cause intracellular pH (pH i ) changes: its diminishing during the first 40-60 min by 0.16-0.18 pH units, then the return to the control level 120 min after irradiation and, finally, the increase by 0.18-0.20 pH units. Simultaneously, the synthesizing activity of the cells changed in the same way. The ATP level changed in the opposite way: increased when pH fell and decreased when pH grew. It was shown that pH i changes were connected with the changes in Na + /H + -exchange system, and they seemed to be primary in the chain of the alterations observed

Two dimensional gel electrophoresis using narrow pH 3-5.6 immobilised pH gradient strips identifies potential novel disease biomarkers in plasma or serum

OpenAIRE

sprotocols

2015-01-01

Authors: Bevin Gangadharan & Nicole Zitzmann ### Abstract Two-dimensional gel electrophoresis (2-DE) is a protein separation technique often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. This protocol describes how to run 2-DE gels using narrow pH 3-5.6 immobilised pH gradient strips to separate 2 mg of serum proteins. pH 3-6 ampholytes are used to enhance the solubility of proteins in this pH range before the serum proteins are separated in the...

Single-cell intracellular nano-pH probes†

Science.gov (United States)

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2016-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

Single-cell intracellular nano-pH probes.

Science.gov (United States)

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

MR imaging of intracellular and extracellular deoxyhemoglobin

International Nuclear Information System (INIS)

Janick, P.A.; Grossman, R.I.; Asakura, T.

1989-01-01

MR imaging was performed on varying concentrations of intracellular and extracellular deoxyhemoglobin as well as varying proportions of deoxyhemoglobin and oxyhemoglobin in vitro at 1.5T with use of standard spin-echo and gradient-refocused spin sequences. This study indicates that susceptibility-induced T2 shortening occurs over a broad range of intracellular deoxyhemoglobin concentrations (maximal at hematocrits between 20% and 45%), reflecting diffusional effects at the cellular level. T2* gradient-echo imaging enhances the observed hypointensity in images of intracellular deoxyhemoglobin. The characteristic MR appearance of acute hemotomas can be modeled by the behavior of intracellular and extracellular deoxyhemoglobin and oxyhemoglobin

Precision sensing by two opposing gradient sensors: how does Escherichia coli find its preferred pH level?

Science.gov (United States)

Hu, Bo; Tu, Yuhai

2013-07-02

It is essential for bacteria to find optimal conditions for their growth and survival. The optimal levels of certain environmental factors (such as pH and temperature) often correspond to some intermediate points of the respective gradients. This requires the ability of bacteria to navigate from both directions toward the optimum location and is distinct from the conventional unidirectional chemotactic strategy. Remarkably, Escherichia coli cells can perform such a precision sensing task in pH taxis by using the same chemotaxis machinery, but with opposite pH responses from two different chemoreceptors (Tar and Tsr). To understand bacterial pH sensing, we developed an Ising-type model for a mixed cluster of opposing receptors based on the push-pull mechanism. Our model can quantitatively explain experimental observations in pH taxis for various mutants and wild-type cells. We show how the preferred pH level depends on the relative abundance of the competing sensors and how the sensory activity regulates the behavioral response. Our model allows us to make quantitative predictions on signal integration of pH and chemoattractant stimuli. Our study reveals two general conditions and a robust push-pull scheme for precision sensing, which should be applicable in other adaptive sensory systems with opposing gradient sensors. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A fluorescent pH probe for acidic organelles in living cells.

Science.gov (United States)

Chen, Jyun-Wei; Chen, Chih-Ming; Chang, Cheng-Chung

2017-09-26

A water-soluble pH sensor, 2-(6-(4-aminostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N, N-dimethylethanamine (ADA), was synthesized based on the molecular design of photoinduced electron transfer (PET) and intramolecular charge transfer (ICT). The fluorescence emission response against a pH value is in the range 3-6, which is suitable for labelling intracellular pH-dependent microenvironments. After biological evolution, ADA is more than a pH biosensor because it is also an endocytosis pathway tracking biosensor that labels endosomes, late endosomes, and lysosome pH gradients. From this, the emissive aggregates of ADA and protonated-ADA in these organs were evaluated to explore how this probe stresses emission colour change to cause these unique cellular images.

Single-cell intracellular nano-pH probes†

OpenAIRE

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular p...

Work-related pain in extrinsic finger extensor musculature of instrumentalists is associated with intracellular pH compartmentation during exercise.

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Angel Moreno-Torres

Full Text Available BACKGROUND: Although non-specific pain in the upper limb muscles of workers engaged in mild repetitive tasks is a common occupational health problem, much is unknown about the associated structural and biochemical changes. In this study, we compared the muscle energy metabolism of the extrinsic finger extensor musculature in instrumentalists suffering from work-related pain with that of healthy control instrumentalists using non-invasive phosphorus magnetic resonance spectroscopy ((31P-MRS. We hypothesize that the affected muscles will show alterations related with an impaired energy metabolism. METHODOLOGY/PRINCIPAL FINDINGS: We studied 19 volunteer instrumentalists (11 subjects with work-related pain affecting the extrinsic finger extensor musculature and 8 healthy controls. We used (31P-MRS to find deviations from the expected metabolic response to exercise in phosphocreatine (PCr, inorganic phosphate (Pi, Pi/PCr ratio and intracellular pH kinetics. We observed a reduced finger extensor exercise tolerance in instrumentalists with myalgia, an intracellular pH compartmentation in the form of neutral and acid compartments, as detected by Pi peak splitting in (31P-MRS spectra, predominantly in myalgic muscles, and a strong association of this pattern with the condition. CONCLUSIONS/SIGNIFICANCE: Work-related pain in the finger extrinsic extensor muscles is associated with intracellular pH compartmentation during exercise, non-invasively detectable by (31P-MRS and consistent with the simultaneous energy production by oxidative metabolism and glycolysis. We speculate that a deficit in energy production by oxidative pathways may exist in the affected muscles. Two possible explanations for this would be the partial and/or local reduction of blood supply and the reduction of the muscle oxidative capacity itself.

Work-Related Pain in Extrinsic Finger Extensor Musculature of Instrumentalists Is Associated with Intracellular pH Compartmentation during Exercise

Science.gov (United States)

Moreno-Torres, Angel; Rosset-Llobet, Jaume; Pujol, Jesus; Fàbregas, Sílvia; Gonzalez-de-Suso, Jose-Manuel

2010-01-01

Background Although non-specific pain in the upper limb muscles of workers engaged in mild repetitive tasks is a common occupational health problem, much is unknown about the associated structural and biochemical changes. In this study, we compared the muscle energy metabolism of the extrinsic finger extensor musculature in instrumentalists suffering from work-related pain with that of healthy control instrumentalists using non-invasive phosphorus magnetic resonance spectroscopy (31P-MRS). We hypothesize that the affected muscles will show alterations related with an impaired energy metabolism. Methodology/Principal Findings We studied 19 volunteer instrumentalists (11 subjects with work-related pain affecting the extrinsic finger extensor musculature and 8 healthy controls). We used 31P-MRS to find deviations from the expected metabolic response to exercise in phosphocreatine (PCr), inorganic phosphate (Pi), Pi/PCr ratio and intracellular pH kinetics. We observed a reduced finger extensor exercise tolerance in instrumentalists with myalgia, an intracellular pH compartmentation in the form of neutral and acid compartments, as detected by Pi peak splitting in 31P-MRS spectra, predominantly in myalgic muscles, and a strong association of this pattern with the condition. Conclusions/Significance Work-related pain in the finger extrinsic extensor muscles is associated with intracellular pH compartmentation during exercise, non-invasively detectable by 31P-MRS and consistent with the simultaneous energy production by oxidative metabolism and glycolysis. We speculate that a deficit in energy production by oxidative pathways may exist in the affected muscles. Two possible explanations for this would be the partial and/or local reduction of blood supply and the reduction of the muscle oxidative capacity itself. PMID:20161738

Altered Ca fluxes and contractile state during pH changes in cultured heart cells

International Nuclear Information System (INIS)

Kim, D.; Smith, T.W.

1987-01-01

The authors studied mechanisms underlying changes in myocardial contractile state produced by intracellular (pH/sub i/) or extracellular (pH 0 ) changes in pH using cultured chick embryo ventricular cells. A change in pH 0 of HEPES-buffered medium from 7.4 to 6.0 or to 8.8 changed the amplitude of cell motion by -85 or +60%, and 45 Ca uptake at 10 s by -29 or +22%, respectively. The pH 0 induced change in Ca uptake was not sensitive to nifedipine but was Na gradient dependent. Changes in pH/sub i/ produced by NH 4 Cl or preincubation in media at pH values ranging from 6.0 to 8.8 failed to alter significantly 45 Ca uptake or efflux. However, larger changes in pH/sub i/ were associated with altered Ca uptake. Changes in pH 0 from 7.5 to 6.0 or to 8.8 were associated with initial changes in 45 Ca efflux by +17 or -18%, respectively, and these effects were not Na dependent. Exposure of cells to 20 mM NH 4 Cl produced intracellular alkalinization and a positive inotropic effect, whereas subsequent removal of NH 4 Cl caused intracellular acidification and a negative inotropic effect. There was, however, a lack of close temporal relationships between pH/sub i/ and contractile state. These results indicated that pH 0 -induced changes in contractile state in cultured heart cells are closely correlated with altered transarcolemmal Ca movements and presumably are due to these Ca flux changes

31P MR spectroscopic measurement of intracellular pH in normal human hearts

International Nuclear Information System (INIS)

Kwon, Jae Hyun; Lee, Hui Joong; Jang, Yong Min

2002-01-01

To assess the usefulness of intracellular pH (pHi), calculated by determining the shift of a high-energy metabolite such as inorganic phosphate (Pi) of γ-ATP after performing MRS with ECG-gated two-dimensional 31 P CSI (chemical shift imaging), as a parameter for the overall state of the intracellular milieu. Proto decoupled 31 P CSI was performed on a 1.5-T scanner using a 1 H 31 P dual-tuned surface coil. Cardiac MRS data were obtained from eight normal volunteers aged 24-32 years with no history of heart disease. From the spectra obtained from several regions of the heart, peack position and peak area were estimated. The metabolic ratios of α-, β-, γ-ATP, PCr, Pi, phosphodiester and diphosphoglycerate were calculated, and pHi was estimated from the chemical shift of Pi and γ-ATP resonance. We then compared the data for the anterior myocardium with those previously published. The major phosphorous metabolites identified in these human hearts were as follows: PCr, at -0.1 to +0.1 ppm; three phosphate peaks from ATP, with a chemical shift centered at about -2.7 ppm (γ-ATP), -7.8 ppm (α-ATP), and -16.3 ppm (β-ATP); and phosphodiester (PDE) at 2-3 ppm, inorganic phosphate (Pi) at 4.5-5.4 ppm, and diphosphoglycerate (DPG) at 5.4-6.3 ppm. The PCr/β-ATP ratio was 2.20±0.17 and the PDE/β-ATP ratio, 1.04±0.09 pHi readings were 7.31±0.23 (calculated by the shift of Pi) and 6.81±0.20 (calculated by the shift of γ-ATP). Pi/PCR was 0.539, a ratio higher than that mentioned in previously published reports. The measurement of intracellular metabolism was affected by various kinds of factors. We believe, however, that pHi readings indicate the overall state of the cardiac intracellular milieu. An unexpected pHi readings, seen at MRS, may reflect errors in the MR procedure itself and, or in the analytical method

In situ photo-immobilised pH gradient isoelectric focusing and zone electrophoresis integrated two-dimensional microfluidic chip electrophoresis for protein separation

International Nuclear Information System (INIS)

Lin, Fengmin; Yu, Shiyong; Gu, Le; Zhu, Xuetao; Wang, Jianshe; Zhu, Han; Lu, Yi; Wang, Yihua; Deng, Yulin; Geng, Lina

2015-01-01

A method is introduced for open-column photo-induced site-selective immobilization of pH gradients in a layer of PEG-methacrylate in a multi-dimensional microfluidic chip for use in electrophoresis. It has several attractive features: (a) mixtures of fluorescently labelled proteins carbonic anhydrase, catalase and myoglobin in their native state can be separated by pH-gradient isoelectric focusing (IEF) and zone electrophoresis (CZE) using integrated 2D chip electrophoresis; (b) compared to strip packing or monolithic photo-immobilization, it overcomes the shortcomings of free carrier ampholyte-based 2D chip electrophoresis in an easy way; (c) larger amount of sample can be loaded into the open column-mode electrophoresis (d) immobilized pH gradients can be re-used and the chip can be recycled; (e) a multilayer 3D pH gradient is established by a layer-by-layer assembly technique to further increase the separation capacity. In our perception, this strategy has a large potential in microfluidic chip-based separation schemes because of its simplicity, separation power, re-usability, and separation capacity. (author)

In vivo measurement of intracellular pH in human brain during different tensions of carbon dioxide in arterial blood. A 31P-NMR study

DEFF Research Database (Denmark)

Jensen, K E; Thomsen, C; Henriksen, O

1988-01-01

The effect of changes in carbon dioxide tension in arterial blood upon intracellular pH in brain tissue was studied in seven healthy volunteers, aged 22-45 years. The pH changes were monitored by use of 31P nuclear magnetic resonance spectroscopy, performed on a whole-body 1.5 Tesla Siemens imaging...

Phosphorus nuclear magnetic resonance studies of the energetic state and of the intracellular pH of the isolated rat heart in the course of ischemia

Energy Technology Data Exchange (ETDEWEB)

Rossi, A [Grenoble-1 Univ., 38 (France); Martin, J; de Leiris, J [CEA Centre d' Etudes Nucleaires de Grenoble, 38 (France). Lab. de Chimie Organique Physique

1981-01-01

Continuous measurements of high energy phosphate compounds and intracellular pH in perfused, beating rat hearts, were performed by /sup 31/P nuclear magnetic resonance (NMR). Hearts were placed in a 15 mm NMR tube and perfused at 28/sup 0/C by conventional methods with a phosphate-free solution. Phosphorus NMR spectra were recorded at 101,3 MHz in a Brucker WP 250 spectrometer. Global mild ischemia was achieved by reducing the coronary flow to 1/10 of its initial value. Changes in creatine phosphate (CP) and inorganic phosphate (Pi) levels and intracellular pH (pHi) were monitored in the course of a 50 min ischemia and during the post-ischemic phase. When the breakdown of CP was less than 30%, the decrease in pHi was about 0.1 to 0.2 pH unit; for a greater CP decrease, the fall in pHi was about 1 pH unit.

Effects of 42 deg. C hyperthermia on intracellular pH in ovarian carcinoma cells during acute or chronic exposure to low extracellular pH

International Nuclear Information System (INIS)

Wahl, Miriam L.; Bobyock, Suzanne B.; Leeper, Dennis B.; Owen, Charles S.

1997-01-01

Purpose: To determine whether intracellular pH (pH i ) is affected during hyperthermia in substrate-attached cells and whether acute extracellular acidification potentiates the cytotoxicity of hyperthermia via an effect on pH i . Methods and Materials: The pH i was determined in cells attached to extracellular matrix proteins loaded with the fluorescent indicator dye BCECF at 37 deg. C and during 42 deg. C hyperthermia at an extracellular pH (pH e ) of 6.7 or 7.3 in cells. Effects on pH i during hyperthermia are compared to effects on clonogenic survival after hyperthermia at pH e 7.3 and 6.7 of cells grown at pH e 7.3, or of cells grown and monitored at pH e 6.7. Results: The results show that pH i values are affected by substrate attachments. Cells attached to extracellular matrix proteins had better signal stability, low dye leakage and evidence of homeostatic regulation of pH i during heating. The net decrease in pH i in cells grown and assayed at pH e = 7.3 during 42 deg. C hyperthermia was 0.28 units and the decrease in low pH adapted cells heated at pH e = 6.7 was 0.14 units. Acute acidification from pH e = 7.3 to pH e = 6.7 at 37 deg. C caused an initial reduction of 0.5-0.8 unit in pH i , but a partial recovery followed during the next 60-90 min. Concurrent 42 deg. C hyperthermia caused the same initial reduction in pH i in acutely acidified cells, but inhibited the partial recovery that occurred during the next 60-90 min at 37 deg. C. After 4 h at 37 deg. C, the net change in pH i in acutely acidified cells was 0.30 pH unit, but at 42 deg. C is 0.63 pH units. The net change in pH i correlated inversely with clonogenic survival. Conclusions: Hyperthermia causes a pH i reduction in cells which was smaller in magnitude by 50% in low pH adapted cells. Hyperthermia inhibited the partial recovery from acute acidification that was observed at 37 deg. C in substrate attached cells, in parallel with a lower subsequent clonogenic survival

Leucocins 4010 from Leuconostoc carnosum cause a matrix related decrease in intracellular pH of Listeria monocytogenes

DEFF Research Database (Denmark)

Fang, Weihuan; Budde, Birgitte Bjørn; Siegumfeldt, Henrik

2006-01-01

A mixed culture of single cells of Listeria monocytogenes and the bacteriocin producing Leuconostoc carnosum 4010 showed growth inhibition of L. monocytogenes, although the intracellular pH (pHi) of L. monocytogenes followed by fluorescence ratio imaging microscopy was not affected. Furthermore, L...

A mathematical model for the generation and control of a pH gradient in an immobilized enzyme system involving acid generation.

Science.gov (United States)

Chen, G; Fournier, R L; Varanasi, S

1998-02-20

An optimal pH control technique has been developed for multistep enzymatic synthesis reactions where the optimal pH differs by several units for each step. This technique separates an acidic environment from a basic environment by the hydrolysis of urea within a thin layer of immobilized urease. With this technique, a two-step enzymatic reaction can take place simultaneously, in proximity to each other, and at their respective optimal pH. Because a reaction system involving an acid generation represents a more challenging test of this pH control technique, a number of factors that affect the generation of such a pH gradient are considered in this study. The mathematical model proposed is based on several simplifying assumptions and represents a first attempt to provide an analysis of this complex problem. The results show that, by choosing appropriate parameters, the pH control technique still can generate the desired pH gradient even if there is an acid-generating reaction in the system. Copyright 1998 John Wiley & Sons, Inc.

Regulation of the glutamine transporter SN1 by extracellular pH and intracellular sodium ions

International Nuclear Information System (INIS)

Broeer, A.; Broeer, S.; Setiawan, I.; Lang, F.

2001-01-01

Full text: SN1 has recently been identified as one of the major glutamine transporters in hepatocytes and brain astrocytes. It appears to be the molecular correlate of the system N amino acid transporter. Two different transport mechanisms have been proposed for this transporter. Either an electroneutral mechanism, in which glutamine uptake is coupled to an exchange of 1Na + and 1H + , or an electrogenic mechanism coupled to the exchange of 2Na + against 1H + . This study was performed to solve the discrepancies and to investigate the reversibility of the transporter. When expressed in Xenopus laevis oocytes glutamine uptake activity increased strongly with increasing pH. In agreement with the pH-dependence we found that uptake of glutamine was accompanied by an alkalization of the cytosol, indicating that SN1 mediates Glutamine/H + -Antiport. Uptake of glutamine into oocytes was Na + -dependent. Analysis of the Na + -dependence of glutamine transport and Flux studies using 22 Na + indicated that two or more sodium ions were cotransported together with glutamine. However, at the same time intracellular Na + was exchanged against extracellular Na + . Taken together with the results of the pH-dependence it is proposed that SN1 mediates a Na + /Na + -exchange and a Na + /H + -exchange, both being coupled to the transport of glutamine. In agreement with this mechanism we found that acidic pH caused a reversal of the transporter. To investigate the source of the glutamine-induced inward currents, we compared inward currents generated by the 1Na + /glutamine cotransporter ATA1 with those generated by SN1. Currents induced by glutamine uptake in SN1 expressing oocytes were only a fraction of the currents induced by glutamine in ATA1 expressing oocytes, indicating that they were not generated by a stoichiometric uptake of ions. It is concluded that SN1 is tightly regulated by pH and intracellular Na + -ions and is capable of mediating glutamine uptake and release

Fermentation pH influences the physiological-state dynamics of Lactobacillus bulgaricus CFL1 during pH-controlled culture.

Science.gov (United States)

Rault, Aline; Bouix, Marielle; Béal, Catherine

2009-07-01

This study aims at better understanding the effects of fermentation pH and harvesting time on Lactobacillus bulgaricus CFL1 cellular state in order to improve knowledge of the dynamics of the physiological state and to better manage starter production. The Cinac system and multiparametric flow cytometry were used to characterize and compare the progress of the physiological events that occurred during pH 6 and pH 5 controlled cultures. Acidification activity, membrane damage, enzymatic activity, cellular depolarization, intracellular pH, and pH gradient were determined and compared during growing conditions. Strong differences in the time course of viability, membrane integrity, and acidification activity were displayed between pH 6 and pH 5 cultures. As a main result, the pH 5 control during fermentation allowed the cells to maintain a more robust physiological state, with high viability and stable acidification activity throughout growth, in opposition to a viability decrease and fluctuation of activity at pH 6. This result was mainly explained by differences in lactate concentration in the culture medium and in pH gradient value. The elevated content of the ionic lactate form at high pH values damaged membrane integrity that led to a viability decrease. In contrast, the high pH gradient observed throughout pH 5 cultures was associated with an increased energetic level that helped the cells maintain their physiological state. Such results may benefit industrial starter producers and fermented-product manufacturers by allowing them to better control the quality of their starters, before freezing or before using them for food fermentation.

Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.

Science.gov (United States)

Schewe, Bettina; Schmälzlin, Elmar; Walz, Bernd

2008-03-01

Blowfly salivary gland cells have a vacuolar-type H(+)-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pH(i)) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pH(i) in salivary gland cells is 7.5+/-0.3 pH units (N=96), higher than that expected from passive H(+) distribution; (2) low 5-HT concentrations (0.3-3 nmol l(-1)) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l(-1), causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pH(i); (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl(-)-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na(+)-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pH(i) regulation and 5-HT-induced outward H(+) pumping does not cause an alkalinization of pH(i) because of cytosolic H(+) accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

pH- and ion-sensitive polymers for drug delivery

Science.gov (United States)

Yoshida, Takayuki; Lai, Tsz Chung; Kwon, Glen S; Sako, Kazuhiro

2013-01-01

Introduction Drug delivery systems (DDSs) are important for effective, safe, and convenient administration of drugs. pH- and ion-responsive polymers have been widely employed in DDS for site-specific drug release due to their abilities to exploit specific pH- or ion-gradients in the human body. Areas covered Having pH-sensitivity, cationic polymers can mask the taste of drugs and release drugs in the stomach by responding to gastric low pH. Anionic polymers responsive to intestinal high pH are used for preventing gastric degradation of drug, colon drug delivery and achieving high bioavailability of weak basic drugs. Tumor-targeted DDSs have been developed based on polymers with imidazole groups or poly(β-amino ester) responsive to tumoral low pH. Polymers with pH-sensitive chemical linkages, such as hydrazone, acetal, ortho ester and vinyl ester, pH-sensitive cell-penetrating peptides and cationic polymers undergoing pH-dependent protonation have been studied to utilize the pH gradient along the endocytic pathway for intracellular drug delivery. As ion-sensitive polymers, ion-exchange resins are frequently used for taste-masking, counterion-responsive drug release and sustained drug release. Polymers responding to ions in the saliva and gastrointestinal fluids are also used for controlled drug release in oral drug formulations. Expert opinion Stimuli-responsive DDSs are important for achieving site-specific and controlled drug release; however, intraindividual, interindividual and intercellular variations of pH should be considered when designing DDSs or drug products. Combination of polymers and other components, and deeper understanding of human physiology are important for development of pH- and ion-sensitive polymeric DDS products for patients. PMID:23930949

pH- and ion-sensitive polymers for drug delivery.

Science.gov (United States)

Yoshida, Takayuki; Lai, Tsz Chung; Kwon, Glen S; Sako, Kazuhiro

2013-11-01

Drug delivery systems (DDSs) are important for effective, safe, and convenient administration of drugs. pH- and ion-responsive polymers have been widely employed in DDS for site-specific drug release due to their abilities to exploit specific pH- or ion-gradients in the human body. Having pH-sensitivity, cationic polymers can mask the taste of drugs and release drugs in the stomach by responding to gastric low pH. Anionic polymers responsive to intestinal high pH are used for preventing gastric degradation of drug, colon drug delivery and achieving high bioavailability of weak basic drugs. Tumor-targeted DDSs have been developed based on polymers with imidazole groups or poly(β-amino ester) responsive to tumoral low pH. Polymers with pH-sensitive chemical linkages, such as hydrazone, acetal, ortho ester and vinyl ester, pH-sensitive cell-penetrating peptides and cationic polymers undergoing pH-dependent protonation have been studied to utilize the pH gradient along the endocytic pathway for intracellular drug delivery. As ion-sensitive polymers, ion-exchange resins are frequently used for taste-masking, counterion-responsive drug release and sustained drug release. Polymers responding to ions in the saliva and gastrointestinal fluids are also used for controlled drug release in oral drug formulations. Stimuli-responsive DDSs are important for achieving site-specific and controlled drug release; however, intraindividual, interindividual and intercellular variations of pH should be considered when designing DDSs or drug products. Combination of polymers and other components, and deeper understanding of human physiology are important for development of pH- and ion-sensitive polymeric DDS products for patients.

{sup 31}P MR spectroscopic measurement of intracellular pH in normal human hearts

Energy Technology Data Exchange (ETDEWEB)

Kwon, Jae Hyun; Lee, Hui Joong; Jang, Yong Min [Kyungpook National Univ., Taegu (Korea, Republic of)] [and others

2002-05-01

To assess the usefulness of intracellular pH (pHi), calculated by determining the shift of a high-energy metabolite such as inorganic phosphate (Pi) of {gamma}-ATP after performing MRS with ECG-gated two-dimensional {sup 31}P CSI (chemical shift imaging), as a parameter for the overall state of the intracellular milieu. Proto decoupled {sup 31}P CSI was performed on a 1.5-T scanner using a {sup 1}H{sup 31}P dual-tuned surface coil. Cardiac MRS data were obtained from eight normal volunteers aged 24-32 years with no history of heart disease. From the spectra obtained from several regions of the heart, peack position and peak area were estimated. The metabolic ratios of {alpha}-, {beta}-, {gamma}-ATP, PCr, Pi, phosphodiester and diphosphoglycerate were calculated, and pHi was estimated from the chemical shift of Pi and {gamma}-ATP resonance. We then compared the data for the anterior myocardium with those previously published. The major phosphorous metabolites identified in these human hearts were as follows: PCr, at -0.1 to +0.1 ppm; three phosphate peaks from ATP, with a chemical shift centered at about -2.7 ppm ({gamma}-ATP), -7.8 ppm ({alpha}-ATP), and -16.3 ppm ({beta}-ATP); and phosphodiester (PDE) at 2-3 ppm, inorganic phosphate (Pi) at 4.5-5.4 ppm, and diphosphoglycerate (DPG) at 5.4-6.3 ppm. The PCr/{beta}-ATP ratio was 2.20{+-}0.17 and the PDE/{beta}-ATP ratio, 1.04{+-}0.09 pHi readings were 7.31{+-}0.23 (calculated by the shift of Pi) and 6.81{+-}0.20 (calculated by the shift of {gamma}-ATP). Pi/PCR was 0.539, a ratio higher than that mentioned in previously published reports. The measurement of intracellular metabolism was affected by various kinds of factors. We believe, however, that pHi readings indicate the overall state of the cardiac intracellular milieu. An unexpected pHi readings, seen at MRS, may reflect errors in the MR procedure itself and, or in the analytical method.

Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

Science.gov (United States)

Shartau, Ryan B; Crossley, Dane A; Kohl, Zachary F; Brauner, Colin J

2016-07-01

The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2  and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general. © 2016. Published by The Company of Biologists Ltd.

A low cytotoxic and ratiometric fluorescent nanosensor based on carbon-dots for intracellular pH sensing and mapping

International Nuclear Information System (INIS)

Du Fangkai; Ming Yunhao; Zeng Fang; Yu Changmin; Wu Shuizhu

2013-01-01

Intracellular pH plays a critical role in the function of cells, and its regulation is essential for most cellular processes. In this study, we demonstrate a fluorescence resonance energy transfer (FRET)-based ratiometric pH nanosensor with carbon-dot (CD) as the carrier. The sensor was prepared by covalently linking a pH-sensitive fluorescent dye (fluorescein isothiocyanate, FITC) onto carbon-dot. As the FRET donor, the carbon-dot exhibits bright fluorescence emission as well as λ ex -dependent photoluminescence emission, and a suitable excitation wavelength for the donor (CD) can be chosen to match the energy acceptor (fluorescein moiety). The fluorescein moieties on a CD undergo structural and spectral conversion as the pH changes, affording the nanoplatform a FRET-based pH sensor. The CD-based system exhibits a significant change in fluorescence intensity ratio between pH 4 and 8 with a pK a value of 5.69. It also displays excellent water dispersibility, good spectral reversibility, satisfactory cell permeability and low cytotoxicity. Following the living cell uptake, this nanoplatform with dual-chromatic emissions can facilitate real-time visualization of the pH evolution involved in the endocytic pathway of the nanosensor. This reversible and low cytotoxic fluorescent nanoplatform may be highly valuable in a variety of biological studies, such as endocytic trafficking, endosome/lysosome maturation, and pH regulation in subcellular organelles. (paper)

Influence of intracellular acidosis on contractile function in the working rat heart

International Nuclear Information System (INIS)

Jeffrey, F.M.H.; Malloy, C.R.; Radda, G.K.

1987-01-01

The decrease in myocardial contractility during ischemia, hypoxia, and extracellular acidosis has been attributed to intracellular acidosis. Previous studies of the relationship between pH and contractile state have utilized respiratory or metabolic acidosis to alter intracellular pH. The authors developed a model in the working perfused rat heart to study the effects of intracellular acidosis with normal external pH and optimal O 2 delivery. Intracellular pH and high-energy phosphates were monitored by 31 P nuclear magnetic resonance spectroscopy. Hearts were perfused to a steady state with a medium containing 10 mM NH 4 Cl. Acidosis induced a substantial decrease in aortic flow and stroke volume which was associated with little change in peak systolic pressure. It was concluded that (1) for the same intracellular acidosis the influence on tension development was more pronounced with a combined extra- and intracellular acidosis than with an isolated intracellular acidosis, and (2) stroke volume at constant preload was impaired by intracellular acidosis even though changes in developed pressure were minimal. These observations suggest that isolated intracellular acidosis has adverse effects on diastolic compliance and/or relaxation

Isoelectric focusing of dansylated amino acids in immobilized pH gradients

Science.gov (United States)

Bianchi-Bosisio, Adriana; Righetti, Pier Giorgio; Egen, Ned B.; Bier, Milan

1986-01-01

The 21 free amino acids commonly encountered in proteins have been transformed into 'carrier ampholyte' species by reacting their primary amino groups with dansyl chloride. These derivatives can thus be focused in an immobilized pH gradient covering the pH interval 3.1 to 4.1, except for arginine, which still retains a pI of 8.8. Due to their inherent fluorescence, the dansyl derivatives are revealed in UV light, with a sensitivity of the order of 2-4 ng/sq mm. All nearest neighbors are separated except for the following couples: Asn-Gln, Gly-Thr, Val-Ile and Cys-Cys2, with a resolving power, in a Delta(pI) scale, of the order of 0.0018 pH units. Except for a few cases (notably the aromatic amino acids), the order of pI values is well correlated with the pK values of carboxyl groups, suggesting that the latter are not altered by dansylation. From the set of pK(COOH)-pI values of the different amino acids, the pK of the tertiary amino group in the dansyl label has been calculated to be 5.11 + or - 0.06. Knowing the pK of the amino-dansyl and the pI of the excess, free dansyl label (pI = 3.34), a pK of 1.57 is derived for its sulfonic acid group.

The ecology of acidification and recovery: changes in herbivore-algal food web linkages across a stream pH gradient

International Nuclear Information System (INIS)

Ledger, M.E.; Hildrew, A.G.

2005-01-01

We examined the effects of acidification on herbivore-algal food web linkages in headwater streams. We determined the structure and abundance of consumer and benthic algal assemblages, and gauged herbivory, in 10 streams along a pH gradient (mean annual pH 4.6-6.4). Biofilm taxonomic composition changed with pH but total abundance did not vary systematically across the gradient. Mayflies and chironomids dominated under circumneutral conditions but declined with increasing acidity and their consumption of algae was strongly reduced. Contrary to expectations, several putative shredder species consumed algae, maintaining the herbivore-algal linkage where specialist grazers could not persist. These shifts in functioning could render the communities of acidified streams resistant to reinvasion when acidity ameliorates and water chemistry is restored to a pre-acidification condition. This hypothesis is discussed in the light of recent trends in the chemistry and biology of the UK Acid Waters Monitoring Network sites. - Generalist invertebrates maintain algae-herbivore interactions in acid streams

The ecology of acidification and recovery: changes in herbivore-algal food web linkages across a stream pH gradient

Energy Technology Data Exchange (ETDEWEB)

Ledger, M.E. [Queen Mary, University of London, Mile End Road, London E1 4NS (United Kingdom)]. E-mail: m.e.ledger@bham.ac.uk; Hildrew, A.G. [Queen Mary, University of London, Mile End Road, London E1 4NS (United Kingdom)

2005-09-15

We examined the effects of acidification on herbivore-algal food web linkages in headwater streams. We determined the structure and abundance of consumer and benthic algal assemblages, and gauged herbivory, in 10 streams along a pH gradient (mean annual pH 4.6-6.4). Biofilm taxonomic composition changed with pH but total abundance did not vary systematically across the gradient. Mayflies and chironomids dominated under circumneutral conditions but declined with increasing acidity and their consumption of algae was strongly reduced. Contrary to expectations, several putative shredder species consumed algae, maintaining the herbivore-algal linkage where specialist grazers could not persist. These shifts in functioning could render the communities of acidified streams resistant to reinvasion when acidity ameliorates and water chemistry is restored to a pre-acidification condition. This hypothesis is discussed in the light of recent trends in the chemistry and biology of the UK Acid Waters Monitoring Network sites. - Generalist invertebrates maintain algae-herbivore interactions in acid streams.

Alkaline pH sensor molecules.

Science.gov (United States)

Murayama, Takashi; Maruyama, Ichiro N

2015-11-01

Animals can survive only within a narrow pH range. This requires continual monitoring of environmental and body-fluid pH. Although a variety of acidic pH sensor molecules have been reported, alkaline pH sensor function is not well understood. This Review describes neuronal alkaline pH sensors, grouped according to whether they monitor extracellular or intracellular alkaline pH. Extracellular sensors include the receptor-type guanylyl cyclase, the insulin receptor-related receptor, ligand-gated Cl- channels, connexin hemichannels, two-pore-domain K+ channels, and transient receptor potential (TRP) channels. Intracellular sensors include TRP channels and gap junction channels. Identification of molecular mechanisms underlying alkaline pH sensing is crucial for understanding how animals respond to environmental alkaline pH and how body-fluid pH is maintained within a narrow range. © 2015 Wiley Periodicals, Inc.

Surface sulfonamide modification of poly(N-isopropylacrylamide)-based block copolymer micelles to alter pH and temperature responsive properties for controlled intracellular uptake.

Science.gov (United States)

Cyphert, Erika L; von Recum, Horst A; Yamato, Masayuki; Nakayama, Masamichi

2018-06-01

Two different surface sulfonamide-functionalized poly(N-isopropylacrylamide)-based polymeric micelles were designed as pH-/temperature-responsive vehicles. Both sulfadimethoxine- and sulfamethazine-surface functionalized micelles were characterized to determine physicochemical properties, hydrodynamic diameters, zeta potentials, temperature-dependent size changes, and lower critical solution temperatures (LCST) in both pH 7.4 and 6.8 solutions (simulating both physiological and mild low pH conditions), and tested in the incorporation of a proof-of-concept hydrophobic antiproliferative drug, paclitaxel. Cellular uptake studies were conducted using bovine carotid endothelial cells and fluorescently labeled micelles to evaluate if there was enhanced cellular uptake of the micelles in a low pH environment. Both variations of micelles showed enhanced intracellular uptake under mildly acidic (pH 6.8) conditions at temperatures slightly above their LCST and minimal uptake at physiological (pH 7.4) conditions. Due to the less negative zeta potential of the sulfamethazine-surface micelles compared to sulfadimethoxine-surface micelles, and the proximity of their LCST to physiological temperature (37°C), the sulfamethazine variation was deemed more amenable for clinically relevant temperature and pH-stimulated applications. Nevertheless, we believe both polymeric micelle variations have the capacity to be implemented as an intracellular drug or gene delivery system in response to mildly acidic conditions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1552-1560, 2018. © 2018 Wiley Periodicals, Inc.

Photochemical performance of the acidophilic red alga Cyanidium sp. in a pH gradient

Czech Academy of Sciences Publication Activity Database

Kvíderová, Jana

2012-01-01

Roč. 42, č. 2-3 (2012), s. 223-234 ISSN 0169-6149. [European Workshop on Astrobiology of the European- Astrobiology -Network-Association (EANA) /11/. German Aerosp Ctr, Cologne, 11.07.2011-14.07.2011] R&D Projects: GA MŠk 1M0571 Institutional research plan: CEZ:AV0Z60050516 Institutional support: RVO:67985939 Keywords : acidophilic red alga * pH gradient * photochemistry Subject RIV: EF - Botanics Impact factor: 1.831, year: 2012

31P NMR studies of pH homeostasis in intact adult Fasciola hepatica

NARCIS (Netherlands)

Tielens, A.G.M.; Nicolaij, K.; Bergh, van S.G.

1982-01-01

31P NMR was used to measure the intracellular pH in live adult Fasciola hepatica. The results demonstrate that at external pH values above 7.0, pH homeostasis keeps the intracellular pH at 7.0. At external pH values below 7.0 the intracellular pH is less strictly regulated.

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

Science.gov (United States)

Papa, S; Lorusso, M; Izzo, G; Capuano, F

1981-02-15

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi

pH-sensitive intracellular photoluminescence of carbon nanotube-fluorescein conjugates in human ovarian cancer cells

International Nuclear Information System (INIS)

Chen, M T; Ishikawa, F N; Gundersen, M A; Gomez, L M; Vernier, P T; Zhou, C

2009-01-01

To add to the understanding of the properties of functionalized carbon nanotubes in biological applications, we report a monotonic pH sensitivity of the intracellular fluorescence emission of single-walled carbon nanotube-fluorescein carbazide (SWCNT-FC) conjugates in human ovarian cancer cells. Light-stimulated intracellular hydrolysis of the amide linkage and localized intracellular pH changes are proposed as mechanisms. SWCNT-FC conjugates may serve as intracellular pH sensors.

Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2.

Science.gov (United States)

Shirmanova, Marina V; Druzhkova, Irina N; Lukina, Maria M; Matlashov, Mikhail E; Belousov, Vsevolod V; Snopova, Ludmila B; Prodanetz, Natalia N; Dudenkova, Varvara V; Lukyanov, Sergey A; Zagaynova, Elena V

2015-09-01

Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2. A HeLa Kyoto cell line stably expressing SypHer2 in the cytoplasm was used, to perform ratiometric (dual excitation) imaging of the probe in cell culture, in 3D tumor spheroids and in tumor xenografts in living mice. Using SypHer2, pHi was demonstrated to be 7.34±0.11 in monolayer HeLa cells in vitro under standard cultivation conditions. An increasing pHi gradient from the center to the periphery of the spheroids was displayed. We obtained fluorescence ratio maps for HeLa tumors in vivo and ex vivo. Comparison of the map with the pathomorphology and with hypoxia staining of the tumors revealed a correspondence of the zones with higher pHi to the necrotic and hypoxic areas. Our results demonstrate that pHi imaging with the genetically encoded pHi indicator, SypHer2, can be a valuable tool for evaluating tumor progression in xenograft models. We have demonstrated, for the first time, the possibility of using the genetically encoded sensor SypHer2 for ratiometric pH imaging in cancer cells in vitro and in tumors in vivo. SypHer2 shows great promise as an instrument for pHi monitoring able to provide high accuracy and spatiotemporal resolution. Copyright © 2015 Elsevier B.V. All rights reserved.

pH Sensing and Regulation in Cancer

OpenAIRE

Mehdi eDamaghi; Jonathan W. Wojtkowiak; Robert J. Gillies

2013-01-01

Cells maintain intracellular pH (pHi) within a narrow range (7.1-7.2) by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the intracellular pH, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs) and proton-sensing G-protein coupled receptors (GPCRs...

Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.

Directory of Open Access Journals (Sweden)

Ann L Wozniak

2010-09-01

Full Text Available The hepatitis C virus (HCV p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H(+ conductance in vesicles and was able to rapidly equilibrate H(+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5 vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H(+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H(+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection

Monitoring of the Proton Electrochemical Gradient in Reconstituted Vesicles: Quantitative Measurements of Both Transmembrane Potential and Intravesicular pH by Ratiometric Fluorescent Probes

Czech Academy of Sciences Publication Activity Database

Holoubek, A.; Večeř, J.; Sigler, Karel

2007-01-01

Roč. 17, - (2007), s. 201-213 ISSN 1053-0509 Institutional research plan: CEZ:AV0Z50200510 Keywords : transmembrane potential * intracellular ph * oxonol dyes Subject RIV: EE - Microbiology, Virology Impact factor: 2.101, year: 2007

Polymeric gel nanoparticle pH sensors for intracellular measurements

DEFF Research Database (Denmark)

Almdal, Kristoffer; Andresen, Thomas Lars; Benjaminsen, Rikke Vicki

pH range is approximately 4 pH units and thus a nanoparticle sensor with two pH sensitive fluorophores is appropriate. With one pH sensitive fluorophore the output from the sensor follows R=R0+R1/10(pKa-pH), where R is the ratio of fluorescence for the two fluorophores, R0 is the minimum value of R...

Intracellular pH regulation by acid-base transporters in mammalian neurons

Science.gov (United States)

Ruffin, Vernon A.; Salameh, Ahlam I.; Boron, Walter F.; Parker, Mark D.

2014-01-01

Intracellular pH (pHi) regulation in the brain is important in both physiological and physiopathological conditions because changes in pHi generally result in altered neuronal excitability. In this review, we will cover 4 major areas: (1) The effect of pHi on cellular processes in the brain, including channel activity and neuronal excitability. (2) pHi homeostasis and how it is determined by the balance between rates of acid loading (JL) and extrusion (JE). The balance between JE and JL determine steady-state pHi, as well as the ability of the cell to defend pHi in the face of extracellular acid-base disturbances (e.g., metabolic acidosis). (3) The properties and importance of members of the SLC4 and SLC9 families of acid-base transporters expressed in the brain that contribute to JL (namely the Cl-HCO3 exchanger AE3) and JE (the Na-H exchangers NHE1, NHE3, and NHE5 as well as the Na+- coupled HCO3− transporters NBCe1, NBCn1, NDCBE, and NBCn2). (4) The effect of acid-base disturbances on neuronal function and the roles of acid-base transporters in defending neuronal pHi under physiopathologic conditions. PMID:24592239

A pH and solvent optimized reverse-phase ion-paring-LC–MS/MS method that leverages multiple scan-types for targeted absolute quantification of intracellular metabolites

DEFF Research Database (Denmark)

McCloskey, Douglas; Gangoiti, Jon A.; Palsson, Bernhard O.

2015-01-01

Comprehensive knowledge of intracellular biochemistry is needed to accurately understand, model, and manipulate metabolism for industrial and therapeutic applications. Quantitative metabolomics has been driven by advances in analytical instrumentation and can add valuable knowledge to the underst......Comprehensive knowledge of intracellular biochemistry is needed to accurately understand, model, and manipulate metabolism for industrial and therapeutic applications. Quantitative metabolomics has been driven by advances in analytical instrumentation and can add valuable knowledge...... existing reverse phase ion-paring liquid chromatography methods for separation and detection of polar and anionic compounds that comprise key nodes of intracellular metabolism by optimizing pH and solvent composition. In addition, the presented method utilizes multiple scan types provided by hybrid...

Intracellular pH and its response to CO2-driven seawater acidification in symbiotic versus non-symbiotic coral cells.

Science.gov (United States)

Gibbin, Emma M; Putnam, Hollie M; Davy, Simon K; Gates, Ruth D

2014-06-01

Regulating intracellular pH (pHi) is critical for optimising the metabolic activity of corals, yet the mechanisms involved in pH regulation and the buffering capacity within coral cells are not well understood. Our study investigated how the presence of symbiotic dinoflagellates affects the response of pHi to PCO2-driven seawater acidification in cells isolated from Pocillopora damicornis. Using the fluorescent dye BCECF-AM, in conjunction with confocal microscopy, we simultaneously characterised the pHi response in host coral cells and their dinoflagellate symbionts, in symbiotic and non-symbiotic states under saturating light, with and without the photosynthetic inhibitor DCMU. Each treatment was run under control (pH 7.8) and CO2-acidified seawater conditions (decreasing pH from 7.8 to 6.8). After 105 min of CO2 addition, by which time the external pH (pHe) had declined to 6.8, the dinoflagellate symbionts had increased their pHi by 0.5 pH units above control levels when in the absence of DCMU. In contrast, in both symbiotic and non-symbiotic host coral cells, 15 min of CO2 addition (0.2 pH unit drop in pHe) led to cytoplasmic acidosis equivalent to 0.3-0.4 pH units irrespective of whether DCMU was present. Despite further seawater acidification over the duration of the experiment, the pHi of non-symbiotic coral cells did not change, though in host cells containing a symbiont cell the pHi recovered to control levels when photsynthesis was not inhibited. This recovery was negated when cells were incubated with DCMU. Our results reveal that photosynthetic activity of the endosymbiont is tightly coupled with the ability of the host cell to recover from cellular acidosis after exposure to high CO2/low pH. © 2014. Published by The Company of Biologists Ltd.

Quantification of pH gradients and implications in insulator-based dielectrophoresis of biomolecules.

Science.gov (United States)

Gencoglu, Aytug; Camacho-Alanis, Fernanda; Nguyen, Vi Thanh; Nakano, Asuka; Ros, Alexandra; Minerick, Adrienne R

2011-09-01

Direct current (DC) insulator-based dielectrophoretic (iDEP) microdevices have the potential to replace traditional alternating current dielectrophoretic devices for many cellular and biomolecular separation applications. The use of large DC fields suggest that electrode reactions and ion transport mechanisms can become important and impact ion distributions in the nanoliters of fluid in iDEP microchannels. This work tracked natural pH gradient formation in a 100 μm wide, 1 cm-long microchannel under applicable iDEP protein manipulation conditions. Using fluorescence microscopy with the pH-sensitive dye FITC Isomer I and the pH-insensitive dye TRITC as a reference, pH was observed to drop drastically in the microchannels within 1 min in a 3000 V/cm electric field; pH drops were observed in the range of 6-10 min within a 100 V/cm electric field and varied based on the buffer conductivity. To address concerns of dye transport impacting intensity data, electrokinetic mobilities of FITC were carefully examined and found to be (i) toward the anode and (ii) 1 to 2 orders of magnitude smaller than H⁺ transport which is responsible for pH drops from the anode toward the cathode. COMSOL simulations of ion transport showed qualitative agreement with experimental results. The results indicate that pH changes are severe enough and rapid enough to influence the net charge of a protein or cause aggregation during iDEP experiments. The results also elucidate reasonable time periods over which the phosphate buffering capacity can counter increases in H⁺ and OH⁻ for unperturbed iDEP manipulations. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Induction of Intracellular Ca2+ and pH Changes in Sf9 Insect Cells by Rhodojaponin-III, A Natural Botanic Insecticide Isolated from Rhododendron molle

Directory of Open Access Journals (Sweden)

Yan-Bo Zhang

2011-04-01

Full Text Available Many studies on intracellular calcium ([Ca2+]i and intracellular pH (pHi have been carried out due to their importance in regulation of different cellular functions. However, most of the previous studies are focused on human or mammalian cells. The purpose of the present study was to characterize the effect of Rhodojaponin-III (R-III on [Ca2+]i and pHi and the proliferation of Sf9 cells. R-III strongly inhibited Sf9 cells proliferation with a time- and dose-dependent manner. Flow cytometry established that R-III interfered with Sf9 cells division and arrested them in G2/M. By using confocal scanning technique, effects of R-III on intracellular free calcium ([Ca2+]i and intracellular pH (pHi in Sf9 cells were determined. R-III induced a significant dose-dependent (1, 10, 100, 200 μg/mL increase in [Ca2+]i and pHi of Sf9 cells in presence of Ca2+-containing solution (Hanks and an irreversible decrease in the absence of extra cellular Ca2+. We also found that both extra cellular Ca2+ and intracellular Ca2+ stores contributed to the increase of [Ca2+]i, because completely treating Sf9 cells with CdCl2 (5 mM, a Ca2+ channels blocker, R-III (100 μg/mL induced a transient elevation of [Ca2+]i in case of cells either in presence of Ca2+ containing or Ca2+ free solution. In these conditions, pHi showed similar changes with that of [Ca2+]i on the whole. Accordingly, we supposed that there was a certain linkage for change of [Ca2+]i, cell cycle arrest, proliferation inhibition in Sf9 cells induced by R-III.

Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane.

Science.gov (United States)

Roz, Netta; Rehavi, Moshe

2003-06-13

Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.

Quantifying intracellular hydrogen peroxide perturbations in terms of concentration

Directory of Open Access Journals (Sweden)

Beijing K. Huang

2014-01-01

Full Text Available Molecular level, mechanistic understanding of the roles of reactive oxygen species (ROS in a variety of pathological conditions is hindered by the difficulties associated with determining the concentration of various ROS species. Here, we present an approach that converts fold-change in the signal from an intracellular sensor of hydrogen peroxide into changes in absolute concentration. The method uses extracellular additions of peroxide and an improved biochemical measurement of the gradient between extracellular and intracellular peroxide concentrations to calibrate the intracellular sensor. By measuring peroxiredoxin activity, we found that this gradient is 650-fold rather than the 7–10-fold that is widely cited. The resulting calibration is important for understanding the mass-action kinetics of complex networks of redox reactions, and it enables meaningful characterization and comparison of outputs from endogenous peroxide generating tools and therapeutics across studies.

Effect of Cu2+ and pH on intracellular calcium content and lipid peroxidation in winter wheat roots

Directory of Open Access Journals (Sweden)

M. E. Riazanova

2015-06-01

Full Text Available The study investigates the effect of copper ions and pH of external solution on intracellular calcium homeostasis and lipid peroxidation in winter wheat roots. Experiment was carried out with winter wheat. Sterile seeds were germinated in Petri dishes on the filter paper soaked with acetic buffer (pH 4.7 and 6.2 at 20 °Cin the dark for 48 hours. Copper was added as CuSO4. It’s concentrations varied from 0 to 50 µM. The Ca2+-fluorescent dye Fluo-3/AM ester was loaded on 60 hour. Root fluorescence with Fluo-3 loading was detected using X-Cite Series 120 Q unit attached to microscope Olympus BX53 with camera Olympus DP72. Imaging of root cells was achieved after exciting with 488 nm laser and collection of emission signals above 512 nm. Preliminary analysis of the images was performed using software LabSens; brightness (fluorescence intensity analysis was carried out by means of ImageJ. Peroxidation of lipids was determined according to Kumar and Knowles method. It was found that pH of solution had effect on release of calcium from intracellular stores. Low pH provokes an increase of [Ca2+]cyt which may be reaction of roots to acidic medium. Copper induces increase in non-selective permeability of plasma membrane and leads to its faster depolarization. This probably initiates Ca-dependent depolarization channels which are responsible for the influx of calcium from apoplast into the cell. Changing of the membrane permeability may occur due to interaction between Cu2+ ions and Ca-binding sites on plasma membrane or may be due to binding of copper with sulfhydryl groups and increasing of POL. Copper may also damage lipid bilayer and change the activity of some non-selective channels and transporters. Reactive oxygen species which are formed under some types of stress factors, especially the effect of heavy metals, can be activators of Ca-channels. Cu2+ ions rise MDA content and promote the oxidative stress. Low medium pH also induces its

Proton gradients and proton-dependent transport processes in the chloroplast

Directory of Open Access Journals (Sweden)

Ricarda eHöhner

2016-02-01

Full Text Available Proton gradients are fundamental to chloroplast function. Across thylakoid membranes, the light induced proton gradient is essential for ATP synthesis. As a result of proton pumping into the thylakoid lumen, an alkaline stromal pH develops, which is required for full activation of pH-dependent Calvin Benson cycle enzymes. This implies that a pH gradient between the cytosol (pH 7 and the stroma (pH 8 is established upon illumination. To maintain this pH gradient chloroplasts actively extrude protons. More than 30 years ago it was already established that these proton fluxes are electrically counterbalanced by Mg2+, K+ or Cl- fluxes, but only recently the first transport systems that regulate the pH gradient were identified. Notably several (Na+,K+/H+ antiporter systems where identified, that play a role in pH gradient regulation, ion homeostasis, osmoregulation, or coupling of secondary active transport. The established pH gradients are important to drive uptake of essential ions and solutes, but not many transporters involved have been identified to date. In this mini review we summarize the current status in the field and the open questions that need to be addressed in order to understand how pH gradients are maintained, how this is interconnected with other transport processes and what this means for chloroplast function.

Increased NBCn1 expression, Na+/ HCO 3 ? co-transport and intracellular pH in human vascular smooth muscle cells with a risk allele for hypertension

OpenAIRE

Ng, Fu Liang; Boedtkjer, Ebbe; Witkowska, Kate; Ren, Meixia; Zhang, Ruoxin; Tucker, Arthur; Aalkj?r, Christian; Caulfield, Mark J.; Ye, Shu

2017-01-01

Abstract Genome-wide association studies have revealed an association between variation at the SLC4A7 locus and blood pressure. SLC4A7 encodes the electroneutral Na+/ HCO 3 ? co-transporter NBCn1 which regulates intracellular pH (pH i ). We conducted a functional study of variants at this locus in primary cultures of vascular smooth muscle and endothelial cells. In both cell types, we found genotype-dependent differences for rs13082711 in DNA-nuclear protein interactions, where the risk allel...

Compatible intracellular ion composition of the host improves carbon assimilation by zooxanthellae in mutualistic symbioses.

Science.gov (United States)

Seibt, C; Schlichter, D

2001-09-01

Cytosymbiotic algae within the host's plasma are exposed to completely different ionic conditions than microalgae living in the sea. The altered ionic gradients, in particular, could be the reason for higher in hospite carbon assimilation levels. To study the effect of varying extracellular ionic conditions on isolated zooxanthellae, their photosynthetic capacity in pure seawater was compared to that in a test medium in which the concentrations of the major inorganic ions, the pH and the osmolality were adjusted to the conditions measured in the host cytoplasm. In this test medium the ratio between oxygen evolution and carbon fixation was 1.2:1.0; in contrast, zooxanthellae in the hyperionic seawater medium showed a comparatively higher oxygen production (2.6:1.0). These results are attributed to a higher energy demand for ion regulation of the isolated algae in the hyperionic medium. Isolated cytosymbionts in seawater need more energy both for the readjustment to the original intracellular ion concentration within the host cell and also for the maintenance of a much steeper gradient during incubation under hyperionic conditions outside the host. The particular intracellular ion concentration of the host cells could have been a decisive evolutionary factor for the very successful establishment of the mutualistic symbioses between anthozoans and dinoflagellates more than 200 million years ago.

Biocompatible click chemistry enabled compartment-specific pH measurement inside E. coli.

Science.gov (United States)

Yang, Maiyun; Jalloh, Abubakar S; Wei, Wei; Zhao, Jing; Wu, Peng; Chen, Peng R

2014-09-19

Bioorthogonal reactions, especially the Cu(I)-catalysed azide-alkyne cycloaddition, have revolutionized our ability to label and manipulate biomolecules under living conditions. The cytotoxicity of Cu(I) ions, however, has hindered the application of this reaction in the internal space of living cells. By systematically surveying a panel of Cu(I)-stabilizing ligands in promoting protein labelling within the cytoplasm of Escherichia coli, we identify a highly efficient and biocompatible catalyst for intracellular modification of proteins by azide-alkyne cycloaddition. This reaction permits us to conjugate an environment-sensitive fluorophore site specifically onto HdeA, an acid-stress chaperone that adopts pH-dependent conformational changes, in both the periplasm and cytoplasm of E. coli. The resulting protein-fluorophore hybrid pH indicators enable compartment-specific pH measurement to determine the pH gradient across the E. coli cytoplasmic membrane. This construct also allows the measurement of E. coli transmembrane potential, and the determination of the proton motive force across its inner membrane under normal and acid-stress conditions.

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor

Science.gov (United States)

Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

2012-01-01

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots. PMID:22407646

Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor.

Science.gov (United States)

Gjetting, Kisten Sisse Krag; Ytting, Cecilie Karkov; Schulz, Alexander; Fuglsang, Anja Thoe

2012-05-01

Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.

Mapping and identification of interferon gamma-regulated HeLa cell proteins separated by immobilized pH gradient two-dimensional gel electrophoresis

DEFF Research Database (Denmark)

Shaw, A.; Larsen, M.; Roepstorff, P.

1999-01-01

magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system...

Changes in microbial communities associated with the sea anemone Anemonia viridis in a natural pH gradient.

Science.gov (United States)

Meron, Dalit; Buia, Maria-Cristina; Fine, Maoz; Banin, Ehud

2013-02-01

Ocean acidification, resulting from rising atmospheric carbon dioxide concentrations, is a pervasive stressor that can affect many marine organisms and their symbionts. Studies which examine the host physiology and microbial communities have shown a variety of responses to the ocean acidification process. Recently, several studies were conducted based on field experiments, which take place in natural CO(2) vents, exposing the host to natural environmental conditions of varying pH. This study examines the sea anemone Anemonia viridis which is found naturally along the pH gradient in Ischia, Italy, with an aim to characterize whether exposure to pH impacts the holobiont. The physiological parameters of A. viridis (Symbiodinium density, protein, and chlorophyll a+c concentration) and its microbial community were monitored. Although reduction in pH was seen to have had an impact on composition and diversity of associated microbial communities, no significant changes were observed in A. viridis physiology, and no microbial stress indicators (i.e., pathogens, antibacterial activity, etc.) were detected. In light of these results, it appears that elevated CO(2) does not have a negative influence on A. viridis that live naturally in the site. This suggests that natural long-term exposure and dynamic diverse microbial communities may contribute to the acclimation process of the host in a changing pH environment.

Monitoring of internal pH gradients within multi-layer tablets by optical methods and EPR imaging.

Science.gov (United States)

Eisenächer, Friederike; Schädlich, Andreas; Mäder, Karsten

2011-09-30

The high variability of gastrointestinal pH is a general challenge regarding constant release from oral drug delivery systems, especially for ionisable drugs. These drugs often show a pH-dependent solubility and therewith associated intra- and inter-individual variability of emerging drug plasma levels. Several strategies have been investigated with the intention to influence the microenvironmental pH (pH(M)) within solid formulations and therefore achieve pH-independent release profiles. Because of the heterogeneity of solid systems, a precise prediction of the occurring pH(M) is rather difficult. It is therefore important to monitor the pH(M) within the formulations to achieve requested release as well as to minimise pH-dependent degradation processes of the active compound. The purpose of the current study was the analysis of pH(M) gradients within 2- and 3-layer tablets during hydration using 3 different techniques for comparison intensions, in particular a pH indicator dye, fluorescence imaging and EPR imaging. The influence of the presence or absence of pH modifying substances and of an additional lipophilic inter layer on the pH(M) was investigated as well as the variation of matrix forming excipient and buffer pH. The influence of the pH(M) on drug release was analysed as well. In addition, benchtop MRI was accomplished to gain a deeper insight on the hydration and erosion behaviour of 2- and 3-layer tablets. Copyright © 2010 Elsevier B.V. All rights reserved.

Microfluidic device having an immobilized pH gradient and PAGE gels for protein separation and analysis

Science.gov (United States)

Sommer, Gregory J.; Hatch, Anson V.; Singh, Anup K.; Wang, Ying-Chih

2012-12-11

Disclosed is a novel microfluidic device enabling on-chip implementation of a two-dimensional separation methodology. Previously disclosed microscale immobilized pH gradients (IPG) are combined with perpendicular polyacrylamide gel electrophoresis (PAGE) microchannels to achieve orthogonal separations of biological samples. Device modifications enable inclusion of sodium dodecyl sulfate (SDS) in the second dimension. The device can be fabricated to use either continuous IPG gels, or the microscale isoelectric fractionation membranes we have also previously disclosed, for the first dimension. The invention represents the first all-gel two-dimensional separation microdevice, with significantly higher resolution power over existing devices.

Intracellular pH and 42.00 C heat response of CHO cells cultured at pH 6.6

International Nuclear Information System (INIS)

Cook, J.A.; Fox, M.H.

1987-01-01

The authors previously reported that cells under chronic low pH (6.6) conditions have altered thermotolerance. They further characterized both the doubling time (t/sub d/) and the internal pH (pH/sub 1/) of CHO cells continuously cultured at pH 6.6 for times greater than one year. The following differences were noted: 1) A t/sub d/ of 16 hr compared to a t/sub d/ of 12 hr for cells at normal pH (7.3) and a t/sub d/ of 25 hr for the acute low pH cells (pH = 6.6; incubation time = 4 hr). 2) A pH/sub i/ 0.1-0.15 pH units > normal cells and 0.3 pH units > acute low pH cells. 3) Survival at 42.0 0 C which differed from both normal and acute low pH cells. The chronic culture was still quite sensitive to 42.0 0 C treatments during the first 5 hr, but developed tolerance at a higher level than cells under acute low pH conditions. The pH/sub i/ of the chronic culture responded to 42.0 0 C heating in a manner similar to that for acute low pH cells. Whether this culture represents a normal response to long term low pH exposure, or was the response of a mutant population is at the present unknown

Cerebral NMR spectroscopy to study intracellular space in vivo: methodological development for diffusion weighted spectroscopy at short time scale and for pH measurement using 31P detection

International Nuclear Information System (INIS)

Marchadour, Charlotte

2013-01-01

NMR spectroscopy is a unique modality to evaluate intracellular environment in vivo. Indeed observed molecules are specifically intracellular and generally have a biochemistry role and a specific cellular compartmentation. That could be a useful tool to understand cell functioning in their environment. My thesis work consisted in development of new sequence in both diffusion and phosphorus NMR spectroscopy.My first study was to develop a diffusion-weighted spectroscopy at ultra-short diffusion time to look at the anomalous diffusion in the rat brain. ADC evolution as a function of time shows that brain metabolites motion is mainly due to random diffusion and that active transport (if exist) are negligible. Data modeling evidences that diffusion at short diffusion time is sensitive to cytoplasm viscosity and short scale crowding. In collaboration with the pharmaceutical company, this technique was chosen to follow up transgenic mice (rTg4510), model of tau pathology. Preliminary results show significant differences of ADC at an early stage of neuro-degenerescence (3 and 6 months).Phosphorus spectroscopy allows observation of metabolites directly implicated in energetic processes. During this thesis, localization sequences were developed to measure intracellular pH in the primate striatum. These sequences are supposed to be used to evaluate the potential of pH as a bio-marker of neuro-degenerescence in a phenotypic model of the Huntington disease in the non-human primate. (author) [fr

Intracellular pH determination by a 31P-NMR technique. The second dissociation constant of phosphoric acid in a biological system.

Science.gov (United States)

Seo, Y; Murakami, M; Watari, H; Imai, Y; Yoshizaki, K; Nishikawa, H; Morimoto, T

1983-09-01

To evaluate the accuracy of pH determination by 31P-NMR, factors which influence the pK value of phosphate were appraised on the basis of the titration of 1 mM phosphate buffer solution. When the method is used for the determination of cytoplasmic pH, ionic strength is the major factor causing shifts of apparent pK (pK') value, and the magnitude of the shift can be predicted from the ionic strength calculated by means of the Debye-Hückel equation. Ions (Na+, K+, Mg2+, and Ca2+) and salivary protein affected the pK' value by 0.1 to 0.3 units in solution with a given ionic strength depending on the species of ion. The form of the titration curve varied with temperature. Based on these results, the value of 6.75 was obtained with the uncertainty of 0.12 for the intracellular pK' of frog muscle at 24 degrees C.

Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

Science.gov (United States)

Gao, Junyuan; Sun, Xiurong; White, Thomas W.; Delamere, Nicholas A.; Mathias, Richard T.

2015-01-01

In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity. PMID:26536260

Toxicity of fatty acid 18:5n3 from Gymnodinium cf. mikimotoi: II. Intracellular pH and K+ uptake in isolated trout hepatocytes.

Science.gov (United States)

Fossat, B; Porthé-Nibelle, J; Sola, F; Masoni, A; Gentien, P; Bodennec, G

1999-01-01

Effects of octadecapentaenoic acid 18:5n3 and other related polyunsaturated fatty acids present in gymnodinium cf. mikimotoi were tested in isolated trout hepatocytes. These exotoxins decreased intracellular pH followed by a slow recovery to initial value and alkalinization of acidic compartments, suggesting an inhibition of vacuolar H(+)-ATPases. Moreover, addition of 18:5n3 to the extracellular medium induced a decrease of K+ uptake into hepatocytes as a result of Na,K-ATPase inhibition. However, high concentrations (10(-5)-10(-3) M) are necessary to induce these effects.

pH homeostasis in Escherichia coli: measurement by 31P nuclear magnetic resonance of methylphosphonate and phosphate

International Nuclear Information System (INIS)

Slonczewski, J.L.; Rosen, B.P.; Alger, J.R.; Macnab, R.M.

1981-01-01

The intracellular pH of Escherichia coli cells, respiring on endogenous energy sources, was monitored continuously by 31 P NMR over an extracellular pH range between 5.5 and 9. pH homeostasis was found to be good over the entire range, with the data conforming to the simple relationship intracellular pH = 7.6 + 0.1(external pH - 7.6) so that the extreme values observed for intracellular pH were 7.4 and 7.8 external pH 5.5 and 9, respectively. As well as inorganic phosphate, we employed the pH-sensitive NMR probe methylphosphonate, which was taken up by glycerol-grown cells and was nontoxic; its pK/sub a/ of 7.65 made it an ideal probe for measurement of cytoplasmic pH and alkaline external pH

Intra- and extracellular pH of the brain in vivo studied by 31P-NMR during hyper- and hypocapnia

DEFF Research Database (Denmark)

Portman, M A; Lassen, N A; Cooper, T G

1991-01-01

Studies were performed to determine the pH relationships among the extracellular, intracellular, and arterial blood compartments in the brain in vivo. Resolution of the extracellular monophosphate resonance peak from the intracellular peak in 31P nuclear magnetic resonance (NMR) spectra of sheep...... brain with the calvarium intact enabled pH measurement in these respective compartments. Sheep were then subjected to both hyper- and hypoventilation, which resulted in a wide range of arterial PCO2 and pH values. Linear regression analysis of pH in these compartments yielded slopes of 0.56 +/- 0.......05 for extracellular pH (pHe) vs. arterial pH, 0.43 +/- 0.078 for intracellular pH (pHi) vs. pHe, and 0.23 +/- 0.056 for pHi vs. arterial pH. These data indicate that CO2 buffering capacity is different and decreases from the intracellular to extracellular to arterial blood compartments. Separation...

Control of red cell volume and pH in trout: Effects of isoproterenol, transport inhibitors, and extracellular pH in bicarbonate/carbon dioxide-buffered media

DEFF Research Database (Denmark)

NIKINMAA, M; STEFFENSEN, JF; TUFTS, BL

1987-01-01

The effects of extracellular pH and beta-adrenergic stimula-tion on the volume and pH of rainbow. trout red cells were studied in HCO3-/ CO2 butfered media. A decrease in extracellular pH caused an increase in red cell volume and a decrease in intracellular pH. The pH-induced changes in cell volume......, and that the Na+/H+ exchanger is not activated by changes in intracellular pH alone. The adrenergic drug, isoproterenol, promoted cell swelling and proton extrusion even in the presence of 10 mM HCO3-, showing that the adrenergic response plays a significant role in the control of cytoplasmic pH. These responses...... were enhanced by a decrease in extracellular pH, showing that the adrenergic response is of benefit to stressed animals. DIDS markedly enhanced the effect of isoproterenol on the pHi, but abolished the increase in red cell volume. The effects of furosemide were similar to those of DIDS, suggesting...

Intracellular pH-sensing using core/shell silica nanoparticles.

Science.gov (United States)

Korzeniowska, B; Woolley, R; DeCourcey, J; Wencel, D; Loscher, C E; McDonagh, C

2014-07-01

An in-depth understanding of biochemical processes occurring within biological systems is key for early diagnosis of disease and identification of appropriate treatments. Nanobiophotonics offers huge potential benefits for intracellular diagnostics and therapeutics. Intracellular sensing using fluorescent nanoparticles is a potentially useful tool for real-time, in vivo monitoring of important cellular analytes. This work is focused on synthesis of optical chemical nanosensors for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly silica matrix with co-encapsulated Texas Red, acting as a reference dye, and pH-sensitive fluorescein isothiocyanate enabling ratiometric quantitative environmental detection. In order to obtain silica-based nanoparticles -70 nm in size, a modified sol-gel-based Stöber method was employed. The potential of these nanosensors for intracellular pH monitoring is demonstrated inside a live human embryonic kidney cell line whereby a significant change in fluorescence is observed when the cell pH is switched from acidic to basic. High loading efficiencies of nanoparticles into the cells is seen, with little effect on cell morphology even following extended nanoparticle exposure (up to 72 h). Nanoparticle incubation time and the fast response of the nanosensor (-2 s) make it a very powerful tool in monitoring the processes occurring within the cytosol.

Relevance of intracellular polarity to accuracy of eukaryotic chemotaxis

International Nuclear Information System (INIS)

Hiraiwa, Tetsuya; Nishikawa, Masatoshi; Shibata, Tatsuo; Nagamatsu, Akihiro; Akuzawa, Naohiro

2014-01-01

Eukaryotic chemotaxis is usually mediated by intracellular signals that tend to localize at the front or back of the cell. Such intracellular polarities frequently require no extracellular guidance cues, indicating that spontaneous polarization occurs in the signal network. Spontaneous polarization activity is considered relevant to the persistent motions in random cell migrations and chemotaxis. In this study, we propose a theoretical model that connects spontaneous intracellular polarity and motile ability in a chemoattractant solution. We demonstrate that the intracellular polarity can enhance the accuracy of chemotaxis. Chemotactic accuracy should also depend on chemoattractant concentration through the concentration-dependent correlation time in the polarity direction. Both the polarity correlation time and the chemotactic accuracy depend on the degree of responsiveness to the chemical gradient. We show that optimally accurate chemotaxis occurs at an intermediate responsiveness of intracellular polarity. Experimentally, we find that the persistence time of randomly migrating Dictyostelium cells depends on the chemoattractant concentration, as predicted by our theory. At the optimum responsiveness, this ameboid cell can enhance its chemotactic accuracy tenfold. (paper)

Dependence of mitochondrial coenzyme A uptake on the membrane electrical gradient

International Nuclear Information System (INIS)

Tahiliani, A.G.

1989-01-01

Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining [3H]CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH

Neuronal pH regulation

DEFF Research Database (Denmark)

Vorstrup, S; Jensen, K E; Thomsen, C

1989-01-01

The intracellular pH in the brain was studied in six healthy volunteers before and immediately after the administration of 2 g of acetazolamide. Phosphorus-31 nuclear magnetic resonance spectroscopy by a 1.5 tesla whole-body scanner was used. The chemical shift between the inorganic phosphate...

Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

Science.gov (United States)

Payne, Christine

2014-03-01

Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

Proteolytische activiteit bij neutrale pH in rundermilt

NARCIS (Netherlands)

Marrink, Jan

1969-01-01

Intracellular enzymes, hydrolysing proteins optimally at acid pH values (acid proteases), have been studied in detail by several investigators. The existence of proteolytic activity at neutral pH in animal tissue extracts, on the other hand, has often been mentioned, but with few reports on the

Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

Science.gov (United States)

Namkoong, Eun; Shin, Yong-Hwan; Bae, Jun-Seok; Choi, Seulki; Kim, Minkyoung; Kim, Nahyun; Hwang, Sung-Min; Park, Kyungpyo

2015-01-01

Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.

Role of Sodium Bicarbonate Cotransporters in Intracellular pH Regulation and Their Regulatory Mechanisms in Human Submandibular Glands.

Directory of Open Access Journals (Sweden)

Eun Namkoong

Full Text Available Sodium bicarbonate cotransporters (NBCs are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1 and electrogenic NBC (NBCe1, with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs and HSG cells. Intracellular pH (pHi was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.

Nanoparticle/Polymer assembled microcapsules with pH sensing property.

Science.gov (United States)

Zhang, Pan; Song, Xiaoxue; Tong, Weijun; Gao, Changyou

2014-10-01

The dual-labeled microcapsules via nanoparticle/polymer assembly based on polyamine-salt aggregates can be fabricated for the ratiometric intracellular pH sensing. After deposition of SiO2 nanoparticles on the poly(allylamine hydrochloride)/multivalent anionic salt aggregates followed by silicic acid treatment, the generated microcapsules are stable in a wide pH range (3.0 ∼ 8.0). pH sensitive dye and pH insensitive dye are simultaneously labeled on the capsules, which enable the ratiometric pH sensing. Due to the rough and positively charged surface, the microcapsules can be internalized by several kinds of cells naturally. Real-time measurement of intracellular pH in several living cells shows that the capsules are all located in acidic organelles after being taken up. Furthermore, the negatively charged DNA and dyes can be easily encapsulated into the capsules via charge interaction. The microcapsules with combination of localized pH sensing and drug loading abilities have many advantages, such as following the real-time transportation and processing of the carriers in cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intracellular Hyper-Acidification Potentiated by Hydrogen Sulfide Mediates Invasive and Therapy Resistant Cancer Cell Death

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Zheng-Wei Lee

2017-10-01

Full Text Available Slow and continuous release of H2S by GYY4137 has previously been demonstrated to kill cancer cells by increasing glycolysis and impairing anion exchanger and sodium/proton exchanger activity. This action is specific for cancer cells. The resulting lactate overproduction and defective pH homeostasis bring about intracellular acidification-induced cancer cell death. The present study investigated the potency of H2S released by GYY4137 against invasive and radio- as well as chemo-resistant cancers, known to be glycolytically active. We characterized and utilized cancer cell line pairs of various organ origins, based on their aggressive behaviors, and assessed their response to GYY4137. We compared glycolytic activity, via lactate production, and intracellular pH of each cancer cell line pair after exposure to H2S. Invasive and therapy resistant cancers, collectively termed aggressive cancers, are receptive to H2S-mediated cytotoxicity, albeit at a higher concentration of GYY4137 donor. While lactate production was enhanced, intracellular pH of aggressive cancers was only modestly decreased. Inherently, the magnitude of intracellular pH decrease is a key determinant for cancer cell sensitivity to H2S. We demonstrated the utility of coupling GYY4137 with either simvastatin, known to inhibit monocarboxylate transporter 4 (MCT4, or metformin, to further boost glycolysis, in bringing about cell death for aggressive cancers. Simvastatin inhibiting lactate extrusion thence contained excess lactate induced by GYY4137 within intracellular compartment. In contrast, the combined exposure to both GYY4137 and metformin overwhelms cancer cells with lactate over-production exceeding its expulsion rate. Together, GYY4137 and simvastatin or metformin synergize to induce intracellular hyper-acidification-mediated cancer cell death.

Effect of external pH on the cytoplasmic and vacuolar pHs in Mung bean root-tip cells

International Nuclear Information System (INIS)

Torimitsu, Keiichi; Yazaki, Yoshiaki; Nagasuka, Kinuyo; Ohta, Eiji; Sakata, Makoto

1984-01-01

The effect of the external pH on the intracellular pH in mung bean (Vigna mungo (L.) Hepper) root-tip cells was investigated with the 31 P nuclear magnetic resonance (NMR) method. The 31 P NMR spectra showed three peaks caused by cytoplasmic G-6-P, cytoplasmic Psub(i) and vacuolar Psub(i). The cytoplasmic and vacuolar pHs could be determined by comparing the Psub(i) chemical shifts with the titration curve. When the external pH was changed over a range from pH 3 to 10, the cytoplasmic pH showed smaller changes than the vacuolar pH, suggesting that the former is regulated more strictly than the latter. The H + -ATPase inhibitor, DCCD, caused the breakdown of the mechanism that regulates the intracellular pH. H + -ATPase appears to have an important part in the regulation of the intracellular pH. (author)

Role of Cl−–HCO3 − exchanger AE3 in intracellular pH homeostasis in cultured murine hippocampal neurons, and in crosstalk to adjacent astrocytes

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Salameh, Ahlam I.; Hübner, Christian A.

2016-01-01

Key points A polymorphism of human AE3 is associated with idiopathic generalized epilepsy. Knockout of AE3 in mice lowers the threshold for triggering epileptic seizures. The explanations for these effects are elusive.Comparisons of cells from wild‐type vs. AE3–/– mice show that AE3 (present in hippocampal neurons, not astrocytes; mediates HCO3 – efflux) enhances intracellular pH (pHi) recovery (decrease) from alkali loads in neurons and, surprisingly, adjacent astrocytes.During metabolic acidosis (MAc), AE3 speeds initial acidification, but limits the extent of pHi decrease in neurons and astrocytes.AE3 speeds re‐alkalization after removal of MAc in neurons and astrocytes, and speeds neuronal pHi recovery from an ammonium prepulse‐induced acid load.We propose that neuronal AE3 indirectly increases acid extrusion in (a) neurons via Cl– loading, and (b) astrocytes by somehow enhancing NBCe1 (major acid extruder). The latter would enhance depolarization‐induced alkalinization of astrocytes, and extracellular acidification, and thereby reduce susceptibility to epileptic seizures. Abstract The anion exchanger AE3, expressed in hippocampal (HC) neurons but not astrocytes, contributes to intracellular pH (pHi) regulation by facilitating the exchange of extracellular Cl– for intracellular HCO3 –. The human AE3 polymorphism A867D is associated with idiopathic generalized epilepsy. Moreover, AE3 knockout (AE3–/–) mice are more susceptible to epileptic seizure. The mechanism of these effects has been unclear because the starting pHi in AE3–/– and wild‐type neurons is indistinguishable. The purpose of the present study was to use AE3–/– mice to investigate the role of AE3 in pHi homeostasis in HC neurons, co‐cultured with astrocytes. We find that the presence of AE3 increases the acidification rate constant during pHi recovery from intracellular alkaline loads imposed by reducing [CO2]. The presence of AE3 also speeds intracellular

Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids.

Science.gov (United States)

Treyer, Andrea; Mateus, André; Wiśniewski, Jacek R; Boriss, Hinnerk; Matsson, Pär; Artursson, Per

2018-06-04

Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability ( F ic ) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F ic . The induction of NL did not further increase drug binding but led to altered F ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

Evaluation of differences between dual salt-pH gradient elution and mono gradient elution using a thermodynamic model: Simultaneous separation of six monoclonal antibody charge and size variants on preparative-scale ion exchange chromatographic resin.

Science.gov (United States)

Lee, Yi Feng; Jöhnck, Matthias; Frech, Christian

2018-02-21

The efficiencies of mono gradient elution and dual salt-pH gradient elution for separation of six mAb charge and size variants on a preparative-scale ion exchange chromatographic resin are compared in this study. Results showed that opposite dual salt-pH gradient elution with increasing pH gradient and simultaneously decreasing salt gradient is best suited for the separation of these mAb charge and size variants on Eshmuno ® CPX. Besides giving high binding capacity, this type of opposite dual salt-pH gradient also provides better resolved mAb variant peaks and lower conductivity in the elution pools compared to single pH or salt gradients. To have a mechanistic understanding of the differences in mAb variants retention behaviors of mono pH gradient, parallel dual salt-pH gradient, and opposite dual salt-pH gradient, a linear gradient elution model was used. After determining the model parameters using the linear gradient elution model, 2D plots were used to show the pH and salt dependencies of the reciprocals of distribution coefficient, equilibrium constant, and effective ionic capacity of the mAb variants in these gradient elution systems. Comparison of the 2D plots indicated that the advantage of opposite dual salt-pH gradient system with increasing pH gradient and simultaneously decreasing salt gradient is the noncontinuous increased acceleration of protein migration. Furthermore, the fitted model parameters can be used for the prediction and optimization of mAb variants separation in dual salt-pH gradient and step elution. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Impact of seawater acidification on pH at the tissue–skeleton interface and calcification in reef corals

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Venn, Alexander A.; Tambutté, Eric; Holcomb, Michael; Laurent, Julien; Allemand, Denis; Tambutté, Sylvie

2013-01-01

Insight into the response of reef corals and other major marine calcifiers to ocean acidification is limited by a lack of knowledge about how seawater pH and carbonate chemistry impact the physiological processes that drive biomineralization. Ocean acidification is proposed to reduce calcification rates in corals by causing declines in internal pH at the calcifying tissue–skeleton interface where biomineralization takes place. Here, we performed an in vivo study on how partial-pressure CO2-driven seawater acidification impacts intracellular pH in coral calcifying cells and extracellular pH in the fluid at the tissue–skeleton interface [subcalicoblastic medium (SCM)] in the coral Stylophora pistillata. We also measured calcification in corals grown under the same conditions of seawater acidification by measuring lateral growth of colonies and growth of aragonite crystals under the calcifying tissue. Our findings confirm that seawater acidification decreases pH of the SCM, but this decrease is gradual relative to the surrounding seawater, leading to an increasing pH gradient between the SCM and seawater. Reductions in calcification rate, both at the level of crystals and whole colonies, were only observed in our lowest pH treatment when pH was significantly depressed in the calcifying cells in addition to the SCM. Overall, our findings suggest that reef corals may mitigate the effects of seawater acidification by regulating pH in the SCM, but they also highlight the role of calcifying cell pH homeostasis in determining the response of reef corals to changes in external seawater pH and carbonate chemistry. PMID:23277567

Impact of seawater acidification on pH at the tissue-skeleton interface and calcification in reef corals.

Science.gov (United States)

Venn, Alexander A; Tambutté, Eric; Holcomb, Michael; Laurent, Julien; Allemand, Denis; Tambutté, Sylvie

2013-01-29

Insight into the response of reef corals and other major marine calcifiers to ocean acidification is limited by a lack of knowledge about how seawater pH and carbonate chemistry impact the physiological processes that drive biomineralization. Ocean acidification is proposed to reduce calcification rates in corals by causing declines in internal pH at the calcifying tissue-skeleton interface where biomineralization takes place. Here, we performed an in vivo study on how partial-pressure CO(2)-driven seawater acidification impacts intracellular pH in coral calcifying cells and extracellular pH in the fluid at the tissue-skeleton interface [subcalicoblastic medium (SCM)] in the coral Stylophora pistillata. We also measured calcification in corals grown under the same conditions of seawater acidification by measuring lateral growth of colonies and growth of aragonite crystals under the calcifying tissue. Our findings confirm that seawater acidification decreases pH of the SCM, but this decrease is gradual relative to the surrounding seawater, leading to an increasing pH gradient between the SCM and seawater. Reductions in calcification rate, both at the level of crystals and whole colonies, were only observed in our lowest pH treatment when pH was significantly depressed in the calcifying cells in addition to the SCM. Overall, our findings suggest that reef corals may mitigate the effects of seawater acidification by regulating pH in the SCM, but they also highlight the role of calcifying cell pH homeostasis in determining the response of reef corals to changes in external seawater pH and carbonate chemistry.

The effect of bacteriocin-producing Lactobacillus plantarum strains on the intracellular pH of sessile and planktonic Listeria monocytongenes single cells

DEFF Research Database (Denmark)

Nielsen, Dennis Sandris; Cho, Gyu-Sung; Hanak, Alexander

2010-01-01

and/or bacteriocin-producing LAB as “natural” food preservatives in foods such as cheese, meat and ready-to-eat products. Some strains of Lactobacillus plantarum produce bacteriocins termed plantaricins. Using a single-cell based approach, the effect on the intracellular pH as a measure......A wide range of lactic acid bacteria (LAB) produce bacteriocins mainly active against other closely related LAB, but some bacteriocins are also active against the food-borne pathogen Listeria monocytogenes. With the aim of increasing food safety it has thus been considered to utilise bacteriocins...

Radiation and/or hyperthermia sensitivity of human melanoma cells grown for several days in media with reduced pH

International Nuclear Information System (INIS)

Zoelzer, F.; Streffer, C.

1999-01-01

MeWo cells were used throughout. Cells were incubated for 0, 3 or 6 days in media with pH 6.5, 6.7, 6.9, 7.1 or 7.3. The sensitivity against 250 kV X-rays and hyperthermia at 43 C were determined in the colony-forming assay. The intracellular pH was measured flow cytometrically using 5(and 6)-carboxyfluoroescein. Calibration curves were established with cells incubated in different buffers containing nigericin to equilibrate intra- and extracellular pH. Results: Cell growth was optimal with pH 7.3 and 7.1 in the medium, somewhat reduced at pH 6.9, and largely inhibited at pH 6.7 and 6.5. Radiation and/or hyperthermia sensitivities were noticeably increased after several days of incubation at reduced pH; the surviving fraction after 4 Gy and 1 h at 43 C either alone or in combination being a factor of 2 to 4 lower at pH 6.5 than at 7.3. These changes in sensitivity could not be correlated with changes in the intracellular pH. Cells seemed to be capable of regulating this parameter very well; the flow cytometric measurements revealed that the intracellular pH was 7.2±0.2 irrespective of the extracellular pH in the range considered here. Conclusions: In contrast to the almost generally accepted hypothesis that intracellular pH is decisive for the heat sensitivity, the human melanoma cells studied here became sensitive after a few days of incubation under acidic conditions without changes in the intracellular pH. Other factors seem to be influencing the cellular response to radiation and/or heat under chronically low pH. (orig./MG) [de

Intracellular forms of menadione-dependent small-colony variants of methicillin-resistant Staphylococcus aureus are hypersusceptible to β-lactams in a THP-1 cell model due to cooperation between vacuolar acidic pH and oxidant species.

Science.gov (United States)

Garcia, Laetitia G; Lemaire, Sandrine; Kahl, Barbara C; Becker, Karsten; Proctor, Richard A; Tulkens, Paul M; Van Bambeke, Françoise

2012-12-01

Phagocytosed methicillin-resistant Staphylococcus aureus (MRSA) are susceptible to β-lactams because of an acid-induced conformational change of penicillin-binding protein (PBP) 2a within phagolysosomes. We have examined whether this mechanism applies to menD and hemB small-colony variants (SCVs) of the COL MRSA strain, using cloxacillin, meropenem, doripenem, and vancomycin as comparator. Intracellularly, the change in cfu from post-phagocytosis inoculum was measured after 24 h of incubation with antibiotics combined or not with N-acetylcysteine (NAC; oxidant species scavenger); the relative potency (C(s)) was calculated from the Hill equation of concentration-response curves. Extracellularly, the effect of a pre-incubation with H(2)O(2) was determined on MICs and killing at pH 7.4 and 5.5. Intracellularly, the β-lactam C(s) was similar for the COL strain and the hemB mutant and not modified or slightly decreased (2- to 16-fold) by NAC. In contrast, the C(s) was 100- to 900-fold lower for the menD mutant, but similar to that for the COL strain when NAC was present. Extracellularly, β-lactam MICs were markedly reduced at pH 5.5 for the parental strain and the haemin-supplemented hemB mutant, with limited additional effect of pre-incubation with H(2)O(2). In contrast, MICs remained elevated at pH 5.5 for the menD mutant (supplemented with menadione sodium bisulphite or not), but were 7-10 dilutions lower after pre-incubation with H(2)O(2). Vancomycin MICs were unaltered in all conditions, with no marked effect of NAC on C(s). Cooperation between acidic pH and oxidant species confers high potency to β-lactams against intracellular forms of menD SCVs of MRSA.

pH Sensing and Regulation in Cancer

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Mehdi eDamaghi

2013-12-01

Full Text Available Cells maintain intracellular pH (pHi within a narrow range (7.1-7.2 by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the intracellular pH, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs and proton-sensing G-protein coupled receptors (GPCRs. In this review, we will discuss some of the major players in proton sensing at the plasma membrane and their downstream consequences in cancer cells and how these pH-mediated changes affect processes such as migration and metastasis. The complex mechanisms by which they transduce acid pH signals to the cytoplasm and nucleus are not well understood. However, there is evidence that expression of proton-sensing GPCRs such as GPR4, TDAG8, and OGR1 can regulate aspects of tumorigenesis and invasion, including colfilin and talin regulated actin (de-polymerization. Major mechanisms for maintenance of pHi homeostasis include monocarboxylate, bicarbonate and proton transporters. Notably, there is little evidence suggesting a link between their activities and those of the extracellular H+-sensors, suggesting a mechanistic disconnect between intra- and extra-cellular pH. Understanding the mechanisms of pH sensing and regulation may lead to novel and informed therapeutic strategies that can target acidosis, a common physical hallmark of solid tumors.

Intracellular pH in increased after transformation of Chinese hamster embryo fibroblasts

International Nuclear Information System (INIS)

Ober, S.S.; Pardee, A.B.

1987-01-01

These studies reveal that a series of tumorigenic Chinese hamster embryo fibroblast (CHEF) cell lines maintain an internal pH (pH/sub i/) that is 0.12 +/- 0.04 pH unit above that of the nontumorigenic CHEF/18 parental line. pH measurements were made with [ 14 C]-benzoic acid. This increase of pH/sub i/ in the tumorigenic CHEF cells is not due to autocrine growth factor production or to the persistent activation of pathways previously shown to modulate Na + /H + -antiporter activity present in the CHEF/18 line. These findings suggest that the defect in pH/sub i/ regulation in the tumorigenic CHEF/18 derivatives lies in the Na + /H + antiporter itself. Further studies to determine the biological significance of an increased pH/sub i/ show that the external pH (pH 0 )-dependence curve for initiation of DNA synthesis in the tumorigenic CHEF lines is shifted by approximately 0.2 pH unit toward acidic values relative to that of the nontumorigenic CHEF/18 parent. These data show a critical role for pH/sub i/ in the regulation of DNA synthesis in Chinese hamster embryo fibroblasts and demonstrate that aberrations in pH/sub i/ can contribute to the acquisition of altered growth properties

A chemometric-assisted method for the simultaneous determination of malachite green and crystal violet in water based on absorbance-pH data generated by a homemade pH gradient apparatus.

Science.gov (United States)

Yu, Shuling; Yuan, Xuejie; Yang, Jing; Yuan, Jintao; Shi, Jiahua; Wang, Yali; Chen, Yuewen; Gao, Shufang

2015-01-01

An attractive method of generating second-order data was developed by a dropping technique to generate pH gradient simultaneously coupled with diode-array spectrophotometer scanning. A homemade apparatus designed for the pH gradient. The method and the homemade apparatus were used to simultaneously determine malachite green (MG) and crystal violet (CV) in water samples. The absorbance-pH second-order data of MG or CV were obtained from the spectra of MG or CV in a series of pH values of HCl-KCl solution. The second-order data of mixtures containing MG and CV that coexisted with interferents were analyzed using multidimensional partial least-squares with residual bilinearization. The method and homemade apparatus were used to simultaneously determine MG and CV in fish farming water samples and in river ones with satisfactory results. The presented method and the homemade apparatus could serve as an alternative tool to handle some analysis problems. Copyright © 2015 Elsevier B.V. All rights reserved.

A protein-dye hybrid system as a narrow range tunable intracellular pH sensor.

Science.gov (United States)

Anees, Palapuravan; Sudheesh, Karivachery V; Jayamurthy, Purushothaman; Chandrika, Arunkumar R; Omkumar, Ramakrishnapillai V; Ajayaghosh, Ayyappanpillai

2016-11-18

Accurate monitoring of pH variations inside cells is important for the early diagnosis of diseases such as cancer. Even though a variety of different pH sensors are available, construction of a custom-made sensor array for measuring minute variations in a narrow biological pH window, using easily available constituents, is a challenge. Here we report two-component hybrid sensors derived from a protein and organic dye nanoparticles whose sensitivity range can be tuned by choosing different ratios of the components, to monitor the minute pH variations in a given system. The dye interacts noncovalently with the protein at lower pH and covalently at higher pH, triggering two distinguishable fluorescent signals at 700 and 480 nm, respectively. The pH sensitivity region of the probe can be tuned for every unit of the pH window resulting in custom-made pH sensors. These narrow range tunable pH sensors have been used to monitor pH variations in HeLa cells using the fluorescence imaging technique.

Cross-linked self-assembled micelle based nanosensor for intracellular pH measurements

DEFF Research Database (Denmark)

Ek, Pramod Kumar; Søndergaard, Rikke Vicki; Windschiegl, Barbara

2014-01-01

A micelle based nanosensor was synthesized and investigated as a ratiometric pH sensor for use in measurements in living cells by fluorescent microscopy. The nanosensor synthesis was based on self-assembly of an amphiphilic triblock copolymer, which was chemically cross-linked after micelle......-linked by an amidation reaction using 3,6,9-trioxaundecandioic acid cross-linker. The cross-linked micelle was functionalized with two pH sensitive fluorophores and one reference fluorophore, which resulted in a highly uniform ratiometric pH nanosensor with a diameter of 29 nm. The use of two sensor fluorophores...... provided a sensor with a very broad measurement range that seems to be influenced by the chemical design of the sensor. Cell experiments show that the sensor is capable of monitoring the pH distributions in HeLa cells....

Coral calcifying fluid pH is modulated by seawater carbonate chemistry not solely seawater pH.

Science.gov (United States)

Comeau, S; Tambutté, E; Carpenter, R C; Edmunds, P J; Evensen, N R; Allemand, D; Ferrier-Pagès, C; Tambutté, S; Venn, A A

2017-01-25

Reef coral calcification depends on regulation of pH in the internal calcifying fluid (CF) in which the coral skeleton forms. However, little is known about calcifying fluid pH (pH CF ) regulation, despite its importance in determining the response of corals to ocean acidification. Here, we investigate pH CF in the coral Stylophora pistillata in seawater maintained at constant pH with manipulated carbonate chemistry to alter dissolved inorganic carbon (DIC) concentration, and therefore total alkalinity (A T ). We also investigate the intracellular pH of calcifying cells, photosynthesis, respiration and calcification rates under the same conditions. Our results show that despite constant pH in the surrounding seawater, pH CF is sensitive to shifts in carbonate chemistry associated with changes in [DIC] and [A T ], revealing that seawater pH is not the sole driver of pH CF Notably, when we synthesize our results with published data, we identify linear relationships of pH CF with the seawater [DIC]/[H + ] ratio, [A T ]/ [H + ] ratio and [[Formula: see text

Stair-Step Pattern of Soil Bacterial Diversity Mainly Driven by pH and Vegetation Types Along the Elevational Gradients of Gongga Mountain, China.

Science.gov (United States)

Li, Jiabao; Shen, Zehao; Li, Chaonan; Kou, Yongping; Wang, Yansu; Tu, Bo; Zhang, Shiheng; Li, Xiangzhen

2018-01-01

Ecological understandings of soil bacterial community succession and assembly mechanism along elevational gradients in mountains remain not well understood. Here, by employing the high-throughput sequencing technique, we systematically examined soil bacterial diversity patterns, the driving factors, and community assembly mechanisms along the elevational gradients of 1800-4100 m on Gongga Mountain in China. Soil bacterial diversity showed an extraordinary stair-step pattern along the elevational gradients. There was an abrupt decrease of bacterial diversity between 2600 and 2800 m, while no significant change at either lower (1800-2600 m) or higher (2800-4100 m) elevations, which coincided with the variation in soil pH. In addition, the community structure differed significantly between the lower and higher elevations, which could be primarily attributed to shifts in soil pH and vegetation types. Although there was no direct effect of MAP and MAT on bacterial community structure, our partial least squares path modeling analysis indicated that bacterial communities were indirectly influenced by climate via the effect on vegetation and the derived effect on soil properties. As for bacterial community assembly mechanisms, the null model analysis suggested that environmental filtering played an overwhelming role in the assembly of bacterial communities in this region. In addition, variation partition analysis indicated that, at lower elevations, environmental attributes explained much larger fraction of the β-deviation than spatial attributes, while spatial attributes increased their contributions at higher elevations. Our results highlight the importance of environmental filtering, as well as elevation-related spatial attributes in structuring soil bacterial communities in mountain ecosystems.

Identification of the EcoKI and EcoR1241 Type I Restriction-Modification Enzyme Subunits by Non-Equilibrium pH Gradient Two-Dimensional Gel Electrophoresis

Czech Academy of Sciences Publication Activity Database

Nguyen, L. D.; Cajthamlová, Kamila; Nguyen, H. T.; Weiser, Jaroslav; Holubová, Inge; Weiserová, Marie

2002-01-01

Roč. 47, č. 6 (2002), s. 641-648 ISSN 0015-5632 R&D Projects: GA ČR GA301/00/1369; GA ČR GA204/00/1252 Institutional research plan: CEZ:AV0Z5020903 Keywords : ecoki * non-equilibrium ph * gradient Subject RIV: EE - Microbiology, Virology Impact factor: 0.979, year: 2002

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions*

Science.gov (United States)

Cruz-Gallardo, Isabel; Aroca, Ángeles; Persson, Cecilia; Karlsson, B. Göran; Díaz-Moreno, Irene

2013-01-01

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms. PMID:23902765

Iron Hydroxide Minerals Drive Organic and Phosphorus Chemistry in Subsurface Redox / pH Gradients

Science.gov (United States)

Flores, E.; Barge, L. M.; VanderVelde, D.; Baum, M.

2017-12-01

Iron minerals, particularly iron oxides and oxyhydroxides, are prevalent on Mars and may exist in mixed valence or even reduced states beneath the oxidized surface. Iron (II,III) hydroxides, including green rust, are reactive and potentially catalytic minerals that can absorb and concentrate charged species, while also driving chemical reactions. These minerals are highly redox-sensitive and the presence of organics and/or phosphorus species could affect their mineralogy and/or stability. Conversely, the minerals might be able to drive chemical processes such as amino acid formation, phosphorus oxyanion reactions, or could simply selectively preserve organic species via surface adsorption. In an open aqueous sediment column, soluble products of mineral-driven reactions could also diffuse to sites of different chemical conditions to react even further. We synthesized Fe-hydroxide minerals under various conditions relevant to early Earth and ancient Mars (>3.0 Gyr), anoxically and in the presence of salts likely to have been present in surface or ground waters. Using these minerals we conducted experiments to test whether iron hydroxides could promote amino acid formation, and how the reaction is affected by subsurface gradients of redox, pH, and temperature. We also tested the adsorption of organic and phosphorus species onto Fe-hydroxide minerals at different conditions within the gradients. The suite of organic or phosphorus signatures that may be found in a particular mineral system is a combination of what is synthesized there, what is preferentially concentrated / retained there, and what is preserved against degradation. Further work is needed to determine how these processes could have proceeded on Mars and what mineral-organic signatures, abiotic or otherwise, would be produced from such processes.

A voltage-gated H+ channel underlying pH homeostasis in calcifying coccolithophores.

Directory of Open Access Journals (Sweden)

Alison R Taylor

2011-06-01

Full Text Available Marine coccolithophorid phytoplankton are major producers of biogenic calcite, playing a significant role in the global carbon cycle. Predicting the impacts of ocean acidification on coccolithophore calcification has received much recent attention and requires improved knowledge of cellular calcification mechanisms. Uniquely amongst calcifying organisms, coccolithophores produce calcified scales (coccoliths in an intracellular compartment and secrete them to the cell surface, requiring large transcellular ionic fluxes to support calcification. In particular, intracellular calcite precipitation using HCO₃⁻ as the substrate generates equimolar quantities of H+ that must be rapidly removed to prevent cytoplasmic acidification. We have used electrophysiological approaches to identify a plasma membrane voltage-gated H+ conductance in Coccolithus pelagicus ssp braarudii with remarkably similar biophysical and functional properties to those found in metazoans. We show that both C. pelagicus and Emiliania huxleyi possess homologues of metazoan H(v1 H+ channels, which function as voltage-gated H+ channels when expressed in heterologous systems. Homologues of the coccolithophore H+ channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles for the H(v1 class of proteins. Using single cell imaging, we demonstrate that the coccolithophore H+ conductance mediates rapid H+ efflux and plays an important role in pH homeostasis in calcifying cells. The results demonstrate a novel cellular role for voltage gated H+ channels and provide mechanistic insight into biomineralisation by establishing a direct link between pH homeostasis and calcification. As the coccolithophore H+ conductance is dependent on the trans-membrane H+ electrochemical gradient, this mechanism will be directly impacted by, and may underlie adaptation to, ocean acidification. The presence of this H+ efflux pathway suggests that there is no obligate

Parallel Changes in Intracellular Water Volume and pH Induced by NH3/NH4+ Exposure in Single Neuroblastoma Cells

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Víctor M. Blanco

2013-12-01

Full Text Available Background: Increased blood levels of ammonia (NH3 and ammonium (NH4+, i.e. hyperammonemia, leads to cellular brain edema in humans with acute liver failure. The pathophysiology of this edema is poorly understood. This is partly due to incomplete understanding of the osmotic effects of the pair NH3/NH4+ at the cellular and molecular levels. Cell exposure to solutions containing NH3/NH4+ elicits changes in intracellular pH (pHi, which can in turn affect cell water volume (CWV by activating transport mechanisms that produce net gain or loss of solutes and water. The occurrence of CWV changes caused by NH3/NH4+ has long been suspected, but the mechanisms, magnitude and kinetics of these changes remain unknown. Methods: Using fluorescence imaging microscopy we measured, in real time, parallel changes in pHi and CWV caused by brief exposure to NH3/NH4+ of single cells (N1E-115 neuroblastoma or NG-108 neuroblastoma X glioma loaded with the fluorescent indicator BCECF. Changes in CWV were measured by exciting BCECF at its intracellular isosbestic wavelength (∼438 nm, and pHi was measured ratiometrically. Results: Brief exposure to isosmotic solutions (i.e. having the same osmolality as that of control solutions containing NH4Cl (0.5- 30 mM resulted in a rapid, dose-dependent swelling, followed by isosmotic regulatory volume decrease (iRVD. NH4Cl solutions in which either extracellular [NH3] or [NH4+] was kept constant while the other was changed by varying the pH of the solution, demonstrated that [NH3]o rather than [NH4+]o is the main determinant of the NH4Cl-induced swelling. The iRVD response was sensitive to the anion channel blocker NPPB, and partly dependent on external Ca2+. Upon removal of NH4Cl, cells shrank and displayed isosmotic regulatory volume increase (iRVI. Regulatory volume responses could not be activated by comparable CWV changes produced by anisosmotic solutions, suggesting that membrane stretch or contraction by themselves are

Intracellular pH homeostasis plays a role in the tolerance ofDebaryomyces hansenii and Candida zeylanoides to acidified nitrite

DEFF Research Database (Denmark)

Mortensen, Henrik Dam; Jacobsen, Thomas; Koch, Anette Granly

2008-01-01

. hansenii at an external pH (pHex) value of 4.5 butdid not at pHex 5.5. These results indicate that nitrous acid as such plays an important role in the antifungal effect of acidified nitrite. Furthermore, both yeast species experienced severe growth inhibition and a pHi decrease at pHex 4.5, suggesting...... that at least some of the antifungal effects of acidified nitrite may be due to intracellular acidification. For C. zeylanoides, this phenomenon could be explained in part by the uncoupling effect of energy generation from growth. Debaryomyces hansenii was more tolerant to acidified nitrite at pHex 5.5 than C....... zeylanoides, as determined by the rate of growth initiation. In combination with the fact that D. hansenii was able to maintain pHi homeostasis at pHex 5.5 but C. zeylanoides was not, our results suggest that the ability to maintain pHi homeostasis plays a role in the acidified-nitrite tolerance of D...

Controlling composition and color characteristics of Monascus pigments by pH and nitrogen sources in submerged fermentation.

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Shi, Kan; Song, Da; Chen, Gong; Pistolozzi, Marco; Wu, Zhenqiang; Quan, Lei

2015-08-01

Submerged fermentations of Monascus anka were performed with different nitrogen sources at different pH in 3 L bioreactors. The results revealed that the Monascus pigments dominated by different color components (yellow pigments, orange pigments or red pigments) could be selectively produced through pH control and nitrogen source selection. A large amount of intracellular pigments dominated by orange pigments and a small amount of water-soluble extracellular yellow pigments were produced at low pH (pH 2.5 and 4.0), independently of the nitrogen source employed. At higher pH (pH 6.5), the role of the nitrogen source became more significant. In particular, when ammonium sulfate was used as nitrogen source, the intracellular pigments were dominated by red pigments with a small amount of yellow pigments. Conversely, when peptone was used, intracellular pigments were dominated by yellow pigments with a few red pigments derivatives. Neither the presence of peptone nor ammonium sulfate promoted the production of intracellular orange pigments while extracellular pigments with an orangish red color were observed in both cases, with a higher yield when peptone was used. Two-stage pH control fermentation was then performed to improve desirable pigments yield and further investigate the effect of pH and nitrogen sources on pigments composition. These results provide a useful strategy to produce Monascus pigments with different composition and different color characteristics. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

Science.gov (United States)

Branco, Patrícia; Albergaria, Helena; Arneborg, Nils; Prista, Catarina

2018-05-01

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

The chemistry, physiology and pathology of pH in cancer.

Science.gov (United States)

Swietach, Pawel; Vaughan-Jones, Richard D; Harris, Adrian L; Hulikova, Alzbeta

2014-03-19

Cell survival is conditional on the maintenance of a favourable acid-base balance (pH). Owing to intensive respiratory CO2 and lactic acid production, cancer cells are exposed continuously to large acid-base fluxes, which would disturb pH if uncorrected. The large cellular reservoir of H(+)-binding sites can buffer pH changes but, on its own, is inadequate to regulate intracellular pH. To stabilize intracellular pH at a favourable level, cells control trans-membrane traffic of H(+)-ions (or their chemical equivalents, e.g. ) using specialized transporter proteins sensitive to pH. In poorly perfused tumours, additional diffusion-reaction mechanisms, involving carbonic anhydrase (CA) enzymes, fine-tune control extracellular pH. The ability of H(+)-ions to change the ionization state of proteins underlies the exquisite pH sensitivity of cellular behaviour, including key processes in cancer formation and metastasis (proliferation, cell cycle, transformation, migration). Elevated metabolism, weakened cell-to-capillary diffusive coupling, and adaptations involving H(+)/H(+)-equivalent transporters and extracellular-facing CAs give cancer cells the means to manipulate micro-environmental acidity, a cancer hallmark. Through genetic instability, the cellular apparatus for regulating and sensing pH is able to adapt to extracellular acidity, driving disease progression. The therapeutic potential of disturbing this sequence by targeting H(+)/H(+)-equivalent transporters, buffering or CAs is being investigated, using monoclonal antibodies and small-molecule inhibitors.

A hydrogel based nanosensor with an unprecedented broad sensitivity range for pH measurements in cellular compartments

DEFF Research Database (Denmark)

Zhang, M.; Søndergaard, Rikke Vicki; Ek, Pramod Kumar

2015-01-01

Optical pH nanosensors have been applied for monitoring intracellular pH in real-time for about two decades. However, the pH sensitivity range of most nanosensors is too narrow, and measurements that are on the borderline of this range may not be correct. Furthermore, ratiometric measurements...... of acidic intracellular pH (pH sensor, a fluorophore based nanosensor, with an unprecedented broad measurement range from pH 1.4 to 7.0. In this nanosensor, three p......H-sensitive fluorophores (difluoro-Oregon Green, Oregon Green 488, and fluorescein) and one pH-insensitive fluorophore (Alexa 568) were covalently incorporated into a nanoparticle hydrogel matrix. With this broad range quadruple-labelled nanosensor all physiological relevant pH levels in living cells can be measured...

The enhanced cyan fluorescent protein: a sensitive pH sensor for fluorescence lifetime imaging.

Science.gov (United States)

Poëa-Guyon, Sandrine; Pasquier, Hélène; Mérola, Fabienne; Morel, Nicolas; Erard, Marie

2013-05-01

pH is an important parameter that affects many functions of live cells, from protein structure or function to several crucial steps of their metabolism. Genetically encoded pH sensors based on pH-sensitive fluorescent proteins have been developed and used to monitor the pH of intracellular compartments. The quantitative analysis of pH variations can be performed either by ratiometric or fluorescence lifetime detection. However, most available genetically encoded pH sensors are based on green and yellow fluorescent proteins and are not compatible with multicolor approaches. Taking advantage of the strong pH sensitivity of enhanced cyan fluorescent protein (ECFP), we demonstrate here its suitability as a sensitive pH sensor using fluorescence lifetime imaging. The intracellular ECFP lifetime undergoes large changes (32 %) in the pH 5 to pH 7 range, which allows accurate pH measurements to better than 0.2 pH units. By fusion of ECFP with the granular chromogranin A, we successfully measured the pH in secretory granules of PC12 cells, and we performed a kinetic analysis of intragranular pH variations in living cells exposed to ammonium chloride.

Improvement of the respiration efficiency of Lactococcus lactis by decreasing the culture pH.

Science.gov (United States)

Shi, Weijia; Li, Yu; Gao, Xueling; Fu, Ruiyan

2016-03-01

The growth characteristics and intracellular hemin concentrations of Lactococcus lactis grown under different culture pH and aeration conditions were examined to investigate the effect of culture pH on the respiration efficiency of L. lactis NZ9000 (pZN8148). Cell biomass and biomass yield of L. lactis grown with 4 μg hemin/ml and O2 were higher than those without aeration when the culture pH was controlled at 5-6.5. The culture pH affected the respiratory efficiency in the following order of pH: 5 > 5.5 > 6 > 6.5; the lag phase increased as the culture pH decreased. Hemin accumulation was sensitive to culture pH. Among the four pH conditions, pH 5.5 was optimal for hemin accumulation in the cells. The highest intracellular hemin level in L. lactis resting cells incubated at different pH saline levels (5-6.5) was at pH 5.5. The respiration efficiency of L. lactis under respiration-permissive conditions increases markedly as the culture pH decreases. These results may help develop high cell-density L. lactis cultures. Thus, this microorganism may be used for industrial applications.

pH sensitive quantum dot-anthraquinone nanoconjugates

Science.gov (United States)

Ruedas-Rama, Maria Jose; Hall, Elizabeth A. H.

2014-05-01

Semiconductor quantum dots (QDs) have been shown to be highly sensitive to electron or charge transfer processes, which may alter their optical properties. This feature can be exploited for different sensing applications. Here, we demonstrate that QD-anthraquinone conjugates can function as electron transfer-based pH nanosensors. The attachment of the anthraquinones on the surface of QDs results in the reduction of electron hole recombination, and therefore a quenching of the photoluminescence intensity. For some anthraquinone derivatives tested, the quenching mechanism is simply caused by an electron transfer process from QDs to the anthraquinone, functioning as an electron acceptor. For others, electron transfer and energy transfer (FRET) processes were found. A detailed analysis of the quenching processes for CdSe/ZnS QD of two different sizes is presented. The photoluminescence quenching phenomenon of QDs is consistent with the pH sensitive anthraquinone redox chemistry. The resultant family of pH nanosensors shows pKa ranging ˜5-8, being ideal for applications of pH determination in physiological samples like blood or serum, for intracellular pH determination, and for more acidic cellular compartments such as endosomes and lysosomes. The nanosensors showed high selectivity towards many metal cations, including the most physiologically important cations which exist at high concentration in living cells. The reversibility of the proposed systems was also demonstrated. The nanosensors were applied in the determination of pH in samples mimicking the intracellular environment. Finally, the possibility of incorporating a reference QD to achieve quantitative ratiometric measurements was investigated.

pH sensitive quantum dot–anthraquinone nanoconjugates

International Nuclear Information System (INIS)

Ruedas-Rama, Maria Jose; Hall, Elizabeth A H

2014-01-01

Semiconductor quantum dots (QDs) have been shown to be highly sensitive to electron or charge transfer processes, which may alter their optical properties. This feature can be exploited for different sensing applications. Here, we demonstrate that QD-anthraquinone conjugates can function as electron transfer-based pH nanosensors. The attachment of the anthraquinones on the surface of QDs results in the reduction of electron hole recombination, and therefore a quenching of the photoluminescence intensity. For some anthraquinone derivatives tested, the quenching mechanism is simply caused by an electron transfer process from QDs to the anthraquinone, functioning as an electron acceptor. For others, electron transfer and energy transfer (FRET) processes were found. A detailed analysis of the quenching processes for CdSe/ZnS QD of two different sizes is presented. The photoluminescence quenching phenomenon of QDs is consistent with the pH sensitive anthraquinone redox chemistry. The resultant family of pH nanosensors shows pK a ranging ∼5–8, being ideal for applications of pH determination in physiological samples like blood or serum, for intracellular pH determination, and for more acidic cellular compartments such as endosomes and lysosomes. The nanosensors showed high selectivity towards many metal cations, including the most physiologically important cations which exist at high concentration in living cells. The reversibility of the proposed systems was also demonstrated. The nanosensors were applied in the determination of pH in samples mimicking the intracellular environment. Finally, the possibility of incorporating a reference QD to achieve quantitative ratiometric measurements was investigated

Modeling and simulation of protein elution in linear pH and salt gradients on weak, strong and mixed cation exchange resins applying an extended Donnan ion exchange model.

Science.gov (United States)

Wittkopp, Felix; Peeck, Lars; Hafner, Mathias; Frech, Christian

2018-04-13

Process development and characterization based on mathematic modeling provides several advantages and has been applied more frequently over the last few years. In this work, a Donnan equilibrium ion exchange (DIX) model is applied for modelling and simulation of ion exchange chromatography of a monoclonal antibody in linear chromatography. Four different cation exchange resin prototypes consisting of weak, strong and mixed ligands are characterized using pH and salt gradient elution experiments applying the extended DIX model. The modelling results are compared with the results using a classic stoichiometric displacement model. The Donnan equilibrium model is able to describe all four prototype resins while the stoichiometric displacement model fails for the weak and mixed weak/strong ligands. Finally, in silico chromatogram simulations of pH and pH/salt dual gradients are performed to verify the results and to show the consistency of the developed model. Copyright © 2018 Elsevier B.V. All rights reserved.

pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.

Science.gov (United States)

Zhang, Yunfei; Xie, Qiguang; Robertson, J Brian; Johnson, Carl Hirschie

2012-01-01

We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+) specific; neither Ca(++), Mg(++), Na(+), nor K(+) changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+) ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.

pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.

Directory of Open Access Journals (Sweden)

Yunfei Zhang

Full Text Available We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+ specific; neither Ca(++, Mg(++, Na(+, nor K(+ changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+ ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.

Coordinated regulation of intracellular pH by two glucose-sensing pathways in yeast.

Science.gov (United States)

Isom, Daniel G; Page, Stephani C; Collins, Leonard B; Kapolka, Nicholas J; Taghon, Geoffrey J; Dohlman, Henrik G

2018-02-16

The yeast Saccharomyces cerevisiae employs multiple pathways to coordinate sugar availability and metabolism. Glucose and other sugars are detected by a G protein-coupled receptor, Gpr1, as well as a pair of transporter-like proteins, Rgt2 and Snf3. When glucose is limiting, however, an ATP-driven proton pump (Pma1) is inactivated, leading to a marked decrease in cytoplasmic pH. Here we determine the relative contribution of the two sugar-sensing pathways to pH regulation. Whereas cytoplasmic pH is strongly dependent on glucose abundance and is regulated by both glucose-sensing pathways, ATP is largely unaffected and therefore cannot account for the changes in Pma1 activity. These data suggest that the pH is a second messenger of the glucose-sensing pathways. We show further that different sugars differ in their ability to control cellular acidification, in the manner of inverse agonists. We conclude that the sugar-sensing pathways act via Pma1 to invoke coordinated changes in cellular pH and metabolism. More broadly, our findings support the emerging view that cellular systems have evolved the use of pH signals as a means of adapting to environmental stresses such as those caused by hypoxia, ischemia, and diabetes. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Hyperspectral Imaging Using Intracellular Spies: Quantitative Real-Time Measurement of Intracellular Parameters In Vivo during Interaction of the Pathogenic Fungus Aspergillus fumigatus with Human Monocytes.

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Sara Mohebbi

Full Text Available Hyperspectral imaging (HSI is a technique based on the combination of classical spectroscopy and conventional digital image processing. It is also well suited for the biological assays and quantitative real-time analysis since it provides spectral and spatial data of samples. The method grants detailed information about a sample by recording the entire spectrum in each pixel of the whole image. We applied HSI to quantify the constituent pH variation in a single infected apoptotic monocyte as a model system. Previously, we showed that the human-pathogenic fungus Aspergillus fumigatus conidia interfere with the acidification of phagolysosomes. Here, we extended this finding to monocytes and gained a more detailed analysis of this process. Our data indicate that melanised A. fumigatus conidia have the ability to interfere with apoptosis in human monocytes as they enable the apoptotic cell to recover from mitochondrial acidification and to continue with the cell cycle. We also showed that this ability of A. fumigatus is dependent on the presence of melanin, since a non-pigmented mutant did not stop the progression of apoptosis and consequently, the cell did not recover from the acidic pH. By conducting the current research based on the HSI, we could measure the intracellular pH in an apoptotic infected human monocyte and show the pattern of pH variation during 35 h of measurements. As a conclusion, we showed the importance of melanin for determining the fate of intracellular pH in a single apoptotic cell.

Theoretical predictions of lactate and hydrogen ion distributions in tumours.

Directory of Open Access Journals (Sweden)

Maymona Al-Husari

Full Text Available High levels of lactate and H(+-ions play an important role in the invasive and metastatic cascade of some tumours. We develop a mathematical model of cellular pH regulation focusing on the activity of the Na(+/H(+ exchanger (NHE and the lactate/H(+ symporter (MCT to investigate the spatial correlations of extracellular lactate and H(+-ions. We highlight a crucial role for blood vessel perfusion rates in determining the spatial correlation between these two cations. We also predict critical roles for blood lactate, the activity of the MCTs and NHEs on the direction of the cellular pH gradient in the tumour. We also incorporate experimentally determined heterogeneous distributions of the NHE and MCT transporters. We show that this can give rise to a higher intracellular pH and a lower intracellular lactate but does not affect the direction of the reversed cellular pH gradient or redistribution of protons away from the glycolytic source. On the other hand, including intercellular gap junction communication in our model can give rise to a reversed cellular pH gradient and can influence the levels of pH.

In Vivo determination of intracellular pH using pHLuorin proteins in Fusarium Oxysporum

OpenAIRE

Serrano Salces, Antonio

2016-01-01

Premio extraordinario de Trabajo Fin de Máster curso 2013-2014. Molecular and Cellular, Biotechnology and Genetics. El pH extracelular juega un papel clave en los niveles de fosforilación de las MAP quinasas de Fusarium oxysporum. Además, existen evidencias significativas de que la virulencia de distintos patógenos fúngicos se ve alterada directamente por el pH extracelular. Actualmente se desconoce como el pH extracelular afecta al pH intracelular. En este trabajo, hemos hecho uso de una ...

Active auxin uptake by zucchini membrane vesicles: quantitation using ESR volume and delta pH determinations

International Nuclear Information System (INIS)

Lomax, T.L.; Mehlhorn, R.J.; Briggs, W.R.

1985-01-01

Closed and pH-tight membrane vesicles prepared from hypocotyls of 5-day-old dark-grown seedlings of Cucurbita pepo accumulate the plant growth hormone indole-3-acetic acid along an imposed proton gradient (pH low outside, high inside). The use of electron paramagnetic spin probes permitted quantitation both of apparent vesicle volume and magnitude of the pH gradient. Under the experimental conditions used, hormone accumulation was at minimum 20-fold, a value 4 times larger than what one would predict if accumulation reflected only diffusional equilibrium at the measured pH gradient. It is concluded that hormone uptake is an active process, with each protonated molecule of hormone accompanied by an additional proton. Experiments with ionophores confirm that it is the pH gradient itself which drives the uptake

The evolution of Root effect hemoglobins in the absence of intracellular pH protection of the red blood cell: insights from primitive fishes.

Science.gov (United States)

Regan, Matthew D; Brauner, Colin J

2010-06-01

The Root effect, a reduction in blood oxygen (O(2)) carrying capacity at low pH, is used by many fish species to maximize O(2) delivery to the eye and swimbladder. It is believed to have evolved in the basal actinopterygian lineage of fishes, species that lack the intracellular pH (pH(i)) protection mechanism of more derived species' red blood cells (i.e., adrenergically activated Na(+)/H(+) exchangers; betaNHE). These basal actinopterygians may consequently experience a reduction in blood O(2) carrying capacity, and thus O(2) uptake at the gills, during hypoxia- and exercise-induced generalized blood acidoses. We analyzed the hemoglobins (Hbs) of seven species within this group [American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), mooneye (Hiodon tergisus), and pirarucu (Arapaima gigas)] for their Root effect characteristics so as to test the hypothesis of the Root effect onset pH value being lower than those pH values expected during a generalized acidosis in vivo. Analysis of the haemolysates revealed that, although each of the seven species displayed Root effects (ranging from 7.3 to 40.5% desaturation of Hb with O(2), i.e., Hb O(2) desaturation), the Root effect onset pH values of all species are considerably lower (ranging from pH 5.94 to 7.04) than the maximum blood acidoses that would be expected following hypoxia or exercise (pH(i) 7.15-7.3). Thus, although these primitive fishes possess Hbs with large Root effects and lack any significant red blood cell betaNHE activity, it is unlikely that the possession of a Root effect would impair O(2) uptake at the gills following a generalized acidosis of the blood. As well, it was shown that both maximal Root effect and Root effect onset pH values increased significantly in bowfin over those of the more basal species, toward values of similar magnitude to those of most of the more derived

Gradient-free determination of isoelectric points of proteins on chip.

Science.gov (United States)

Łapińska, Urszula; Saar, Kadi L; Yates, Emma V; Herling, Therese W; Müller, Thomas; Challa, Pavan K; Dobson, Christopher M; Knowles, Tuomas P J

2017-08-30

The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. Here, we introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. To demonstrate the general approachability of this platform, we have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes.

Plant-Sediment Interactions in Salt Marshes - An Optode Imaging Study of O2, pH, and CO 2 Gradients in the Rhizosphere.

Science.gov (United States)

Koop-Jakobsen, Ketil; Mueller, Peter; Meier, Robert J; Liebsch, Gregor; Jensen, Kai

2018-01-01

In many wetland plants, belowground transport of O 2 via aerenchyma tissue and subsequent O 2 loss across root surfaces generates small oxic root zones at depth in the rhizosphere with important consequences for carbon and nutrient cycling. This study demonstrates how roots of the intertidal salt-marsh plant Spartina anglica affect not only O 2 , but also pH and CO 2 dynamics, resulting in distinct gradients of O 2 , pH, and CO 2 in the rhizosphere. A novel planar optode system (VisiSens TD ® , PreSens GmbH) was used for taking high-resolution 2D-images of the O 2 , pH, and CO 2 distribution around roots during alternating light-dark cycles. Belowground sediment oxygenation was detected in the immediate vicinity of the roots, resulting in oxic root zones with a 1.7 mm radius from the root surface. CO 2 accumulated around the roots, reaching a concentration up to threefold higher than the background concentration, and generally affected a larger area within a radius of 12.6 mm from the root surface. This contributed to a lowering of pH by 0.6 units around the roots. The O 2 , pH, and CO 2 distribution was recorded on the same individual roots over diurnal light cycles in order to investigate the interlinkage between sediment oxygenation and CO 2 and pH patterns. In the rhizosphere, oxic root zones showed higher oxygen concentrations during illumination of the aboveground biomass. In darkness, intraspecific differences were observed, where some plants maintained oxic root zones in darkness, while others did not. However, the temporal variation in sediment oxygenation was not reflected in the temporal variations of pH and CO 2 around the roots, which were unaffected by changing light conditions at all times. This demonstrates that plant-mediated sediment oxygenation fueling microbial decomposition and chemical oxidation has limited impact on the dynamics of pH and CO 2 in S. anglica rhizospheres, which may in turn be controlled by other processes such as root

Bioreducible poly(amidoamine)s with charge-reversel properties for intracellular protein delivery

NARCIS (Netherlands)

Coué, G.M.J.P.C.; Engbersen, Johannes F.J.; Hennink, W.E.; Engbersen, J.F.J.

2010-01-01

An effective intracellular protein delivery system was developed using bioreducible disulfide-containing poly(amidoamine)s with negatively charged citraconic side groups that can give charge-reversal upon pH decrease. These water-soluble and linear polymers efficiently self-assemble with proteins

Local adaptations in bryophytes revisited: the genetic structure of the calcium-tolerant peatmoss Sphagnum warnstorfii along geographic and pH gradients

Science.gov (United States)

Mikulášková, Eva; Hájek, Michal; Veleba, Adam; Johnson, Matthew G; Hájek, Tomáš; Shaw, Jonathan A

2015-01-01

Bryophytes dominate some ecosystems despite their extraordinary sensitivity to habitat quality. Nevertheless, some species behave differently across various regions. The existence of local adaptations is questioned by a high dispersal ability, which is thought to redistribute genetic variability among populations. Although Sphagnum warnstorfii is an important ecosystem engineer in fen peatlands, the causes of its rather wide niche along the pH/calcium gradient are poorly understood. Here, we studied the genetic variability of its global populations, with a detailed focus on the wide pH/calcium gradient in Central Europe. Principal coordinates analysis of 12 polymorphic microsatellite loci revealed a significant gradient coinciding with water pH, but independent of geography; even samples from the same fens were clearly separated along this gradient. However, most of the genetic variations remained unexplained, possibly because of the introgression from phylogenetically allied species. This explanation is supported by the small heterogeneous cluster of samples that appeared when populations morphologically transitional to S. subnites, S. rubellum, or S. russowii were included into the analysis. Alternatively, this unexplained variation might be attributed to a legacy of glacial refugia with recently dissolved ecological and biogeographic consequences. Isolation by distance appeared at the smallest scale only (up to 43 km). Negative spatial correlations occurred more frequently, mainly at long distances (up to 950 km), implying a genetic similarity among samples which are very distant geographically. Our results confirm the high dispersal ability of peatmosses, but simultaneously suggested that their ability to cope with a high pH/calcium level is at least partially determined genetically, perhaps via specific physiological mechanisms or a hummock-forming ability. PMID:25628880

Skeletal muscle intracellular pH and levels of high energy phosphates during hypercapnia in intact lizards by 31P NMR

International Nuclear Information System (INIS)

Johnson, D.C.; Hitzig, B.M.; Elmden, K.; McFarland, E.; Koutcher, J.; Kazemi, H.

1986-01-01

Lizards have been shown to reduce ventilation during CO 2 breathing. This is thought to be detrimental to the maintenance of intracellular pH (pHi) and levels of high energy phosphates. The authors subjected chameleons (n=4) to 5% CO 2 breathing and made serial measurements of tail (skeletal) muscle pHi, levels of phosphocreatine (PCr), and ATP utilizing high resolution 31 P NMR. pHi was unchanged from controls (7.27 +/- 0.06 units) (mean +/- SE) during 30 minutes of hypercapnia (7.19 +/- 0.09 units) (p>.2) demonstrating effective regulation of skeletal muscle pHi; however, there were significant decreases in the PCr/ATP ratios to 65% +/- 5% (p 2 availability because there were no increases in the levels of glycolytic intermediates and inorganic phosphate which would indicate tissue hypoxia. It is possible that an active process requiring ATP is required for the maintenance of pHi in the presence of hypercapnia and that the reduction of PCr/ATP ratio is a reflection of an increased utilization of ATP

Determination of the second virial coefficient of bovine serum albumin under varying pH and ionic strength by composition-gradient multi-angle static light scattering.

Science.gov (United States)

Ma, Yingfang; Acosta, Diana M; Whitney, Jon R; Podgornik, Rudolf; Steinmetz, Nicole F; French, Roger H; Parsegian, V Adrian

2015-01-01

Composition-gradient multi-angle static light scattering (CG-MALS) is an emerging technique for the determination of intermolecular interactions via the second virial coefficient B22. With CG-MALS, detailed studies of the second virial coefficient can be carried out more accurately and effectively than with traditional methods. In addition, automated mixing, delivery and measurement enable high speed, continuous, fluctuation-free sample delivery and accurate results. Using CG-MALS we measure the second virial coefficient of bovine serum albumin (BSA) in aqueous solutions at various values of pH and ionic strength of a univalent salt (NaCl). The systematic variation of the second virial coefficient as a function of pH and NaCl strength reveals the net charge change and the isoelectric point of BSA under different solution conditions. The magnitude of the second virial coefficient decreases to 1.13 x 10(-5) ml*mol/g(2) near the isoelectric point of pH 4.6 and 25 mM NaCl. These results illuminate the role of fundamental long-range electrostatic and van der Waals forces in protein-protein interactions, specifically their dependence on pH and ionic strength.

Correlation of acidic and basic carrier ampholyte and immobilized pH gradient two-dimensional gel electrophoresis patterns based on mass spectrometric protein identification

DEFF Research Database (Denmark)

Nawrocki, A; Larsen, Martin Røssel; Podtelejnikov, A V

1998-01-01

Separation of proteins on either carrier ampholyte-based or immobilized pH gradient-based two-dimensional (2-D) gels gives rise to electrophoretic patterns that are difficult to compare visually. In this paper we have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI......-MS) to determine the identities of 335 protein spots in these two 2-D gel systems, including a substantial number of basic proteins which had never been identified before. Proteins that were identified in both gel systems allowed us to cross-reference the gel patterns. Vector analysis of these cross...

Intracellular pH of Mycobacterium avium subsp. paratuberculosis following exposure to antimicrobial compounds monitored at the single cell level

DEFF Research Database (Denmark)

Gaggìa, Francesca; Nielsen, Dennis Sandris; Biavati, Bruno

2010-01-01

for 24h revealed the presence of a subpopulation of cells probably resistant to the antimicrobial compounds tested. Use of nisin and bacteriocin-producing LAB strains could lead to new intervention strategies for the control of MAP based on in vivo application of probiotic cultures as feed additives......Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease; moreover, it seems to be implicated in the development of Crohn's disease in humans. In the present study, fluorescence ratio imaging microscopy (FRIM) was used to assess changes in intracellular pH (p......H(i)) of one strain of MAP after exposure to nisin and neutralized cell-free supernatants (NCSs) from five bacteriocin-producing lactic acid bacteria (LAB) with known probiotic properties. The evaluation of pH(i) by FRIM provides information about the physiological state of bacterial cells, bypassing the long...

Hydrogen production of Enterobacter aerogenes altered by extracellular and intracellular redox states

Energy Technology Data Exchange (ETDEWEB)

Nakashimada, Y.; Rachman, M.A.; Kakizono, T.; Nishio, N. [Hiroshima University, Higashi-Hiroshima (Japan). Graduate School of Advanced Sciences of Matter, Department of Molecular Biotechnology

2002-12-01

Enterobacter aerogenes HU-101, tested for its hydrogen production in batch cultures on various substrates, produced the highest amount of hydrogen when the substrate was glycerol. The yield of hydrogen is a function of the degree to which the substrates are reduced. To examine the effect of intracellular redox state on hydrogen yield, glucose-limiting chemostat cultures were carried out at various pH using strain HU-101 and its mutant AY-2. For both strains, the molar yield and the production rate of hydrogen and the hydrogenase activity in the cell-free extract were optimal at the culture pH of 6.3. The highest NADH/NAD ratio in both strains was also observed at pH 6.3, at which the ratio in AY-2 was more than two-fold that of HU-101. Furthermore, NAD(P)H-dependent hydrogen formation was observed in the cell-free extract of AY-2, and hydrogenase activity was found not in the cytoplasmic but in the cell membrane fraction, suggesting that a high intracellular redox state, that is a high NADH/NAD ratio, would accelerate hydrogen production by driving membrane-bound NAD(P)H-dependent hydrogenase. (author)

Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulation.

Science.gov (United States)

Hayashi, Kentaro; Yamamoto, Takamasa S; Ueno, Naoto

2018-02-05

During the gastrulation stage in animal embryogenesis, the cells leading the axial mesoderm migrate toward the anterior side of the embryo, vigorously extending cell protrusions such as lamellipodia. It is thought that the leading cells sense gradients of chemoattractants emanating from the ectodermal cells and translate them to initiate and maintain the cell movements necessary for gastrulation. However, it is unclear how the extracellular information is converted to the intracellular chemical reactions that lead to motion. Here we demonstrated that intracellular Ca 2+ levels in the protrusion-forming leading cells are markedly higher than those of the following cells and the axial mesoderm cells. We also showed that inhibiting the intracellular Ca 2+ significantly retarded the gastrulation cell movements, while increasing the intracellular Ca 2+ with an ionophore enhanced the migration. We further found that the ionophore treatment increased the active form of the small GTPase Rac1 in these cells. Our results suggest that transient intracellular Ca 2+ signals play an essential role in the active cell migration during gastrulation.

Anomalous pH-Dependent Nanofluidic Salinity Gradient Power.

Science.gov (United States)

Yeh, Li-Hsien; Chen, Fu; Chiou, Yu-Ting; Su, Yen-Shao

2017-12-01

Previous studies on nanofluidic salinity gradient power (NSGP), where energy associated with the salinity gradient can be harvested with ion-selective nanopores, all suggest that nanofluidic devices having higher surface charge density should have higher performance, including osmotic power and conversion efficiency. In this manuscript, this viewpoint is challenged and anomalous counterintuitive pH-dependent NSGP behaviors are reported. For example, with equal pH deviation from its isoelectric point (IEP), the nanopore at pH IEP is shown to have smaller surface charge density but remarkably higher NSGP performance than that at pH > IEP. Moreover, for sufficiently low pH, the NSGP performance decreases with lowering pH (increasing nanopore charge density). As a result, a maximum osmotic power density as high as 5.85 kW m -2 can be generated along with a conversion efficiency of 26.3% achieved for a single alumina nanopore at pH 3.5 under a 1000-fold concentration ratio. Using the rigorous model with considering the surface equilibrium reactions on the pore wall, it is proved that these counterintuitive surface-charge-dependent NSGP behaviors result from the pH-dependent ion concentration polarization effect, which yields the degradation in effective concentration ratio across the nanopore. These findings provide significant insight for the design of next-generation, high-performance NSGP devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Reliable and Non-destructive Method for Monitoring the Stromal pH in Isolated Chloroplasts Using a Fluorescent pH Probe

Directory of Open Access Journals (Sweden)

Pai-Hsiang Su

2017-12-01

Full Text Available The proton gradient established by the pH difference across a biological membrane is essential for many physiological processes, including ATP synthesis and ion and metabolite transport. Currently, ionophores are used to study proton gradients, and determine their importance to biological functions of interest. Because of the lack of an easy method for monitoring the proton gradient across the inner envelope membrane of chloroplasts (ΔpHenv, whether the concentration of ionophores used can effectively abolish the ΔpHenv is not proven for most experiments. To overcome this hindrance, we tried to setup an easy method for real-time monitoring of the stromal pH in buffered, isolated chloroplasts by using fluorescent pH probes; using this method the ΔpHenv can be calculated by subtracting the buffer pH from the measured stromal pH. When three fluorescent dyes, BCECF-AM [2′,7′-bis-(2-carboxyethyl-5-(and-6-carboxyfluorescein acetoxymethyl ester], CFDA-SE [5(6-Carboxyfluorescein diacetate succinimidyl ester] and SNARF-1 carboxylic acid acetate succinimidyl ester were incubated with isolated chloroplasts, BCECF-AM and CFDA-SE, but not the ester-formed SNARF-1 were taken up by chloroplasts and digested with esterase to release high levels of fluorescence. According to its relatively higher pKa value (6.98, near the physiological pH of the stroma, BCECF was chosen for further development. Due to shielding of the excitation and emission lights by chloroplast pigments, the ratiometric fluorescence of BCECF was highly dependent on the concentration of chloroplasts. By using a fixed concentration of chloroplasts, a highly correlated standard curve of pH to the BCECF ratiometric fluorescence with an r-square value of 0.98 was obtained, indicating the reliability of this method. Consistent with previous reports, the light-dependent formation of ΔpHenv can be detected ranging from 0.15 to 0.33 pH units upon illumination. The concentration of the ionophore

The Electrochemical H+ Gradient in the Yeast Rhodotorula glutinis

NARCIS (Netherlands)

Höfer, Milan; Nicolay, Klaas; Robillard, George

1985-01-01

The electrochemical gradient of protons, Δµ~H+, was estimated in the obligatory aerobic yeast Rhodotorula glutinis in the pH0 range from 3 to 8.5. The membrane potential, ΔΨ, was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP+) for negative ΔΨ above pH0

Design, calibration and application of broad-range optical nanosensors for determining intracellular pH

DEFF Research Database (Denmark)

Søndergaard, Rikke Vicki; Henriksen, Jonas Rosager; Andresen, Thomas Lars

2014-01-01

Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis....... This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine...... as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design...

Prediction of the chromatographic retention of acid-base compounds in pH buffered methanol-water mobile phases in gradient mode by a simplified model.

Science.gov (United States)

Andrés, Axel; Rosés, Martí; Bosch, Elisabeth

2015-03-13

Retention of ionizable analytes under gradient elution depends on the pH of the mobile phase, the pKa of the analyte and their evolution along the programmed gradient. In previous work, a model depending on two fitting parameters was recommended because of its very favorable relationship between accuracy and required experimental work. It was developed using acetonitrile as the organic modifier and involves pKa modeling by means of equations that take into account the acidic functional group of the compound (carboxylic acid, protonated amine, etc.). In this work, the two-parameter predicting model is tested and validated using methanol as the organic modifier of the mobile phase and several compounds of higher pharmaceutical relevance and structural complexity as testing analytes. The results have been quite good overall, showing that the predicting model is applicable to a wide variety of acid-base compounds using mobile phases prepared with acetonitrile or methanol. Copyright © 2015 Elsevier B.V. All rights reserved.

pH Regulatory Transporters in Pancreatic Ductal Adenocarcinoma

DEFF Research Database (Denmark)

Kong, Su Chii

The abnormal features of hypoxia and altered metabolisms in solid tumours lead to an increased glycolysis that is uncoupled from oxidative phosphorylation in the TCA cycle. Tumoural cells often exhibit dysregulated expressions and activities of various membrane pH regulatory transporters to cope...... with the elevated acid production from this glycolysis, as well as from cellular ATP hydrolysis, sequentially creating a favourable intracellular pH and hostile acidic tumour microenvironment, fortify the tumour cells with highly invasive, metastatic and drug resistant phenotype. In current work, we study...... proliferation was found to be decreased while apoptosis was increased with concanamycin A treatment, indicative of V-ATPases being involved in PDAC cell survival mechanisms as well. Comprehending pH regulation in tumour cells might provide insights in preventing tumourigenesis by pH disruptions. Data presented...

Dual-layered nanogel-coated hollow lipid/polypeptide conjugate assemblies for potential pH-triggered intracellular drug release.

Directory of Open Access Journals (Sweden)

Wen-Hsuan Chiang

Full Text Available To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid (poly(γ-GA, followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide-g-monomethoxy poly(ethylene glycol in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

Decreased Intracellular pH Induced by Cariporide Differentially Contributes to Human Umbilical Cord-Derived Mesenchymal Stem Cells Differentiation

Directory of Open Access Journals (Sweden)

Wei Gao

2014-01-01

Full Text Available Background/Aims: Na+/H+ exchanger 1 (NHE1 is an important regulator of intracellular pH (pHi. High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. Methods: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs. The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. Results: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, β-catenin expression was up-regulated upon cariporide treatment. Conclusion: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

Bioerosion Accretion Replicate (BAR) data covering in situ calcification and bioerosion rates along pH gradients at two volcanically acidified reefs in Papua New Guinea from 2013-01-18 to 2014-11-10 (NCEI Accession 0156692)

Data.gov (United States)

National Oceanic and Atmospheric Administration, Department of Commerce — Bioerosion Accretion Replicate (BAR) data covering in situ calcification and bioerosion rates along pH gradients at two volcanically acidified reefs in Papua New...

Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

OpenAIRE

Mliki, A; Zimmermann, W

1992-01-01

An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydr...

Plasma membrane Ca²⁺-ATPase isoforms composition regulates cellular pH homeostasis in differentiating PC12 cells in a manner dependent on cytosolic Ca²⁺ elevations

DEFF Research Database (Denmark)

Boczek, Tomasz; Lisek, Malwina; Ferenc, Bozena

2014-01-01

isoforms (PMCA1-4) but only PMCA2 and PMCA3, due to their unique localization and features, perform more specialized function. Using differentiated PC12 cells we assessed the role of PMCA2 and PMCA3 in the regulation of intracellular pH in steady-state conditions and during Ca2+ overload evoked by 59 m....... In steady-state conditions, higher TMRE uptake in PMCA2-knockdown line was driven by plasma membrane potential (Ψp). Nonetheless, mitochondrial membrane potential (Ψm) in this line was dissipated during Ca2+ overload. Cyclosporin and bongkrekic acid prevented Ψm loss suggesting the involvement of Ca2......+-driven opening of mitochondrial permeability transition pore as putative underlying mechanism. The findings presented here demonstrate a crucial role of PMCA2 and PMCA3 in regulation of cellular pH and indicate PMCA membrane composition important for preservation of electrochemical gradient...

Acid-base relations in epithelium of turtle bladder: site of active step in acidification and role of metabolic CO2.

Science.gov (United States)

Steinmetz, P R

1969-07-01

The acid-base relations across the two surfaces of the epithelium of the turtle bladder were examined. By means of the 5,5-dimethyl-2,4-oxazolidinedione (DMO) technique the intracellular OH(-) concentration was measured in the presence and absence of a transepithelial pH gradient. When both sides of the bladder were bathed with solutions free of exogenous CO(2) and bicarbonate at pH 7.41 ([OH(-)] = 239 nmoles/liter), the epithelial cells were alkaline, the mean intracellular [OH(-)] being 347nmoles/liter. This alkalinity of the cells was preserved in bladders that secreted H(+) against a gradient of over 2 pH units. In bathing solutions stirred with 4.85% CO(2) and buffered with 25 mM HCO(3) (-) at pH 7.41 the intracellular [OH(-)] was lower than in CO(2)-free solutions and close to the extracellular [OH(-)]. In the CO(2)-free system anaerobiosis caused increased alkalinity of the cells and inhibition of H(+) secretion presumably by decreased metabolic CO(2) production. Carbonic acid inhibitors reduced H(+) secretion, but had no significant effect on the alkalinity of the cells. An inactive analogue of acetazolamide had no effect on H(+) secretion. The results indicate that the active step in acidification is located near the mucosal surface of the epithelium and that the alkali formed within the epithelial cells moves passively into the serosal solution along an electro-chemical gradient. The inhibitory effect of certain sulfonamides on H(+) secretion by the bladder is directly correlated with their known carbonic anhydrase inhibitory activity, but not associated with a measurable change in the mean intracellular [OH(-)].

Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

Science.gov (United States)

Zhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

2015-04-21

A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

Arginine promotes Proteus mirabilis motility and fitness by contributing to conservation of the proton gradient and proton motive force.

Science.gov (United States)

Armbruster, Chelsie E; Hodges, Steven A; Smith, Sara N; Alteri, Christopher J; Mobley, Harry L T

2014-10-01

Swarming contributes to Proteus mirabilis pathogenicity by facilitating access to the catheterized urinary tract. We previously demonstrated that 0.1-20 mmol/L arginine promotes swarming on normally nonpermissive media and that putrescine biosynthesis is required for arginine-induced swarming. We also previously determined that arginine-induced swarming is pH dependent, indicating that the external proton concentration is critical for arginine-dependent effects on swarming. In this study, we utilized survival at pH 5 and motility as surrogates for measuring changes in the proton gradient (ΔpH) and proton motive force (μH(+) ) in response to arginine. We determined that arginine primarily contributes to ΔpH (and therefore μH(+) ) through the action of arginine decarboxylase (speA), independent of the role of this enzyme in putrescine biosynthesis. In addition to being required for motility, speA also contributed to fitness during infection. In conclusion, consumption of intracellular protons via arginine decarboxylase is one mechanism used by P. mirabilis to conserve ΔpH and μH(+) for motility. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Factors influencing intracellular uptake and radiosensitization by 2-nitroimidazoles in vitro

International Nuclear Information System (INIS)

Brown, D.M.; Gonzalez-Mendez, R.; Brown, J.M.

1983-01-01

In this study it is shown that the radiosensitization of hypoxic Chinese hamster ovary (HA-1) cells in vitro by misonidazole (MIS) and other 1-substituted 2-nitroimidazoles depends on the rate and extent of intracellular uptake of these radiosensitizers, which in turn is governed by their lipophilicity [expressed as the octanol:water partition coefficient (P)]. As the lipophilicity of the compounds decreased, the rate of drug entry into the cells was slower, and below P values of approximately 0.05, peak intracellular drug concentrations were found to be lower than that of MIS (P=0.43). In addition, the number of hydroxyl groups on the side chain of the nitroimidazole molecule influenced the uptake of drug into the cells. For compounds of similar P, but differing in the number of side-chain hydroxyl groups, the addition of a single hydroxyl group to the molecule decreased the amount of drug entering the cell by a factor of approximately 2. These compounds enter the cell by nonmediated passive diffusion since altering the energy (ATP) capacity of the cell by 2-deoxyglucose did not affect uptake. It is also shown that increases in temperature or decreases in pH can increase the intracellular uptake of MIS. For example, equal intracellular and extracellular concentrations (100% uptake) of MIS were obtained if cells were heated to 44-45 0 C for 15 min compared to 20-40% uptake at 37 0 C. Increases in MIS uptake by factors of 2 to 3 could be demonstrated within 30 min when cells were incubated in Hanks' balanced salt solution at pH between 6.0 and 6.3 without loss of cell viability. In addition, MIS uptake in aerobic cultured cells varied from 15 to 60% depending on the cell line and culure conditions used

Differences in functional traits between invasive and native Amaranthus species under simulated acid deposition with a gradient of pH levels

Science.gov (United States)

Wang, Congyan; Wu, Bingde; Jiang, Kun; Zhou, Jiawei

2018-05-01

Co-occurring invasive plant species (invaders hereafter) and natives receive similar or even the same environmental selection pressures. Thus, the differences in functional traits between natives and invaders have become widely recognized as a major driving force of the success of plant invasion. Meanwhile, increasing amounts of acid are deposited into ecosystems. Thus, it is important to elucidate the potential effects of acid deposition on the functional traits of invaders in order to better understand the potential mechanisms for the successful invasion. This study aims to address the differences in functional traits between native red amaranth (Amaranthus tricolor L.; amaranth hereafter) and invasive redroot pigweed (A. retroflexus L.; pigweed hereafter) under simulated acid deposition with a gradient of pH levels. Pigweed was significantly taller than amaranth under most treatments. The greater height of pigweed can lead to greater competitive ability for resource acquisition, particularly for sunlight. Leaf shape index of pigweed was also significantly greater than that of amaranth under all treatments. The greater leaf shape index of pigweed can enhance the efficiency of resource capture (especially sunlight capture) via adjustments to leaf shape and size. Thus, the greater height and leaf shape index of pigweed can significantly enhance its competitive ability, especially under acid deposition. Acid deposition of pH 5.6 significantly increased amaranth leaf width in the co-cultivation due to added nutrients. The pH 4.5 acid deposition treatment significantly increased the specific leaf area of amaranth in the monoculture compared with the pH 5.6 acid deposition treatment and the control. The main mechanism explaining this pattern may be due to acid deposition mediating a hormesis effect on plants, promoting plant growth. The values of the relative competition intensity between amaranth and pigweed for most functional traits were lower than zero under most

Plant-Sediment Interactions in Salt Marshes – An Optode Imaging Study of O2, pH, and CO2 Gradients in the Rhizosphere

Directory of Open Access Journals (Sweden)

Ketil Koop-Jakobsen

2018-05-01

Full Text Available In many wetland plants, belowground transport of O2 via aerenchyma tissue and subsequent O2 loss across root surfaces generates small oxic root zones at depth in the rhizosphere with important consequences for carbon and nutrient cycling. This study demonstrates how roots of the intertidal salt-marsh plant Spartina anglica affect not only O2, but also pH and CO2 dynamics, resulting in distinct gradients of O2, pH, and CO2 in the rhizosphere. A novel planar optode system (VisiSens TD®, PreSens GmbH was used for taking high-resolution 2D-images of the O2, pH, and CO2 distribution around roots during alternating light–dark cycles. Belowground sediment oxygenation was detected in the immediate vicinity of the roots, resulting in oxic root zones with a 1.7 mm radius from the root surface. CO2 accumulated around the roots, reaching a concentration up to threefold higher than the background concentration, and generally affected a larger area within a radius of 12.6 mm from the root surface. This contributed to a lowering of pH by 0.6 units around the roots. The O2, pH, and CO2 distribution was recorded on the same individual roots over diurnal light cycles in order to investigate the interlinkage between sediment oxygenation and CO2 and pH patterns. In the rhizosphere, oxic root zones showed higher oxygen concentrations during illumination of the aboveground biomass. In darkness, intraspecific differences were observed, where some plants maintained oxic root zones in darkness, while others did not. However, the temporal variation in sediment oxygenation was not reflected in the temporal variations of pH and CO2 around the roots, which were unaffected by changing light conditions at all times. This demonstrates that plant-mediated sediment oxygenation fueling microbial decomposition and chemical oxidation has limited impact on the dynamics of pH and CO2 in S. anglica rhizospheres, which may in turn be controlled by other processes such as root

Variations of internal pH in typical Italian sourdough yeasts during co-fermentation with lactobacilli

DEFF Research Database (Denmark)

Valmorri, Sara; Mortensen, Henrik Dam; Jespersen, Lene

2008-01-01

The effects of organic acids (lactic and acetic) and extracellular pH (pHex) on the intracellular pH (pHi) of Saccharomyces cerevisiae and Candida milleri during co-fermentation with lactobacilli were investigated by using Fluorescence-Ratio-Imaging-Microscopy (FRIM). Yeasts were grown in a syste...

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

Science.gov (United States)

Johnson, R G; Pfister, D; Carty, S E; Scarpa, A

1979-11-10

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential

Intracellular ion monitoring using a gold-core polymer-shell nanosensor architecture

Energy Technology Data Exchange (ETDEWEB)

Stanca, S E; Cranfield, C G; Biskup, C [Biomolecular Photonics Group, University Hospital Jena, Teichgraben 8, 07743 Jena (Germany); Nietzsche, S [Centre for Electron Microscopy, University Hospital Jena, Ziegel-muehlenweg 1, 07743 Jena (Germany); Fritzsche, W, E-mail: sarmiza.stanca@mti.uni-jena.de, E-mail: charles.cranfield@mti.uni-jena.de, E-mail: christoph.biskup@mti.uni-jena.de [Institute of Photonic Technology, Albert-Einstein-Strasse 9, 07745 Jena (Germany)

2010-02-05

In this study, we describe the design of new ratiometric fluorescent nanosensors, whose architecture is based on a gold core surrounded by a poly(vinyl alcohol)-polyacetal shell. To the gold core, indicator dyes and reference dyes are attached via a cysteine linker. This nanosensor architecture is flexible with regards to the number and types of fluorophore linkages possible. The robust poly(vinyl alcohol)-polyacetal shell protects the fluorophores linked to the core from non-specific interactions with intracellular proteins. The nanosensors developed in this way are biocompatible and can be easily incorporated into mammalian cells without the use of transfection agents. Here, we show the application of these nanosensors for intracellular pH and sodium ion measurements.

Is central chemoreceptor sensitive to intracellular rather than extracellular pH?

DEFF Research Database (Denmark)

Lassen, N A

1990-01-01

, however, that the elegant studies by Loeschcke & Ahmad have demonstrated that [pH]e and [pH]i are normally tightly and rapidly coupled (Loeschcke & Ahmad, 1980). For this reason, the stimulus might just as well be the intracellular hydrogen ion concentration in the chemoreceptor area. The administration...... the same decrease in [pH]e and the same increase in CBF. In other words CBF acidosis can quantitatively account for the CBF increase induced by acetazolamide. But CO2 and acetazolamide influence [pH]i quite differently, as CO2 drops [pH]i to almost the same extent as [pH]c, while two recent studies by MR...... spectroscopy have shown that acetazolamide does not drop [pH]i measurably, if tissue hypercapnia is prevented in artificially ventilated rabbits or by the mild spontaneous hyperventilation caused by acetazolamide in normal man.(ABSTRACT TRUNCATED AT 250 WORDS)...

pH and Ion Homeostasis on Plant Endomembrane Dynamics: Insights from structural models and mutants of K+/H+ antiporters.

Science.gov (United States)

Sze, Heven; Chanroj, Salil

2018-04-24

Plants remodel their cells through the dynamic endomembrane system. Intracellular pH is important for membrane trafficking, but the determinants of pH homeostasis are poorly defined in plants. Electrogenic proton (H+) pumps depend on counter-ion fluxes to establish transmembrane pH gradients at the plasma membrane and endomembranes. Vacuolar-type H+-ATPase-mediated acidification of the trans-Golgi network (TGN) is crucial for secretion and membrane recycling. Pump and counter-ion fluxes are unlikely to fine-tune pH; rather, alkali cation/H+ antiporters, which can alter pH and/or cation homeostasis locally and transiently, are prime candidates. Plants have a large family of predicted cation/H+ exchangers (CHX) of obscure function, in addition to the well-studied K+(Na+)/H+ exchangers (NHX). Here, we review the regulation of cytosolic and vacuolar pH, highlighting the similarities and distinctions of NHX and CHX members. In planta, alkalinization of the TGN or vacuole by NHXs promotes membrane trafficking, endocytosis, cell expansion, and growth. CHXs localize to endomembranes and/or the plasma membrane, contribute to male fertility, pollen tube guidance, pollen wall construction, stomatal opening, and in soybean (Glycine max), tolerance to salt stress. Three-dimensional structural models and mutagenesis of Arabidopsis thaliana genes have allowed us to infer that AtCHX17 and AtNHX1 share a global architecture and a translocation core like bacterial Na+/H+ antiporters. Yet the presence of distinct residues suggests some CHXs differ from NHXs in pH sensing and electrogenicity. How H+ pumps, counter-ion fluxes, and cation/H+ antiporters are linked with signaling and membrane trafficking to remodel membranes and cell walls awaits further investigation. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Regulation of neuronal pH by the metabotropic Zn(2+)-sensing Gq-coupled receptor, mZnR/GPR39.

Science.gov (United States)

Ganay, Thibault; Asraf, Hila; Aizenman, Elias; Bogdanovic, Milos; Sekler, Israel; Hershfinkel, Michal

2015-12-01

Synaptically released Zn(2+) acts as a neurotransmitter, in part, by activating the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39). In previous work using epithelial cells, we described crosstalk between Zn(2+) signaling and changes in intracellular pH and/or extracellular pH (pHe). As pH changes accompany neuronal activity under physiological and pathological conditions, we tested whether Zn(2+) signaling is involved in regulation of neuronal pH. Here, we report that up-regulation of a major H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), is induced by mZnR/GPR39 activation in an extracellular-regulated kinase 1/2-dependent manner in hippocampal neurons in vitro. We also observed that changes in pHe can modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. Similarly, Zn(2+)-dependent extracellular-regulated kinase 1/2 phosphorylation and up-regulation of NHE activity were absent at acidic pHe. Thus, our results suggest that when pHe is maintained within the physiological range, mZnR/GPR39 activation can up-regulate NHE-dependent recovery from intracellular acidification. During acidosis, as pHe drops, mZnR/GPR39-dependent NHE activation is inhibited, thereby attenuating further H(+) extrusion. This mechanism may serve to protect neurons from excessive decreases in pHe. Thus, mZnR/GPR39 signaling provides a homeostatic adaptive process for regulation of intracellular and extracellular pH changes in the brain. We show that the postsynaptic metabotropic Zn(2+)-sensing Gq protein-coupled receptor (mZnR/GPR39) activation induces up-regulation of a major neuronal H(+) extrusion pathway, the Na(+)/H(+) exchanger (NHE), thereby enhancing neuronal recovery from intracellular acidification. Changes in extracellular pH (pHe), however, modulate neuronal mZnR/GPR39-dependent signaling, resulting in reduced activity at pHe 8 or 6.5. This mechanism may serve to protect neurons from excessive

Well-Defined Poly(Ortho Ester Amides) for Potential Drug Carriers: Probing the Effect of Extra- and Intracellular Drug Release on Chemotherapeutic Efficacy.

Science.gov (United States)

Yan, Guoqing; Wang, Jun; Qin, Jiejie; Hu, Liefeng; Zhang, Panpan; Wang, Xin; Tang, Rupei

2017-07-01

To compare the chemotherapeutic efficacy determined by extra- and intracellular drug release strategies, poly(ortho ester amide)-based drug carriers (POEAd-C) with well-defined main-chain lengths, are successfully constructed by a facile method. POEAd-C3-doxorubicin (DOX) can be rapidly dissolved to release drug at tumoral extracellular pH (6.5-7.2), while POEAd-C6-DOX can rapidly release drug following gradual swelling at intracellular pH (5.0-6.0). In vitro cytotoxicity shows that POEAd-C3-DOX exhibits more toxic effect on tumor cells than POEAd-C6-DOX at extracellular pH, but POEAd-C6-DOX has stronger tumor penetration and inhibition in vitro and in vivo tumor models. So, POEAd-C6-DOX with the intracellular drug release strategy has stronger overall chemotherapeutic efficacy than POEAd-C3-DOX with extracellular drug release strategy. It is envisioned that these poly(ortho ester amides) can have great potential as drug carriers for efficient chemotherapy with further optimization. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of Peroxisomal pH in Living Mammalian Cells Using pHRed.

Science.gov (United States)

Godinho, Luis F; Schrader, Michael

2017-01-01

Organelle pH homeostasis is crucial for maintaining proper cellular function. The nature of the peroxisomal pH remains somewhat controversial, with several studies reporting conflicting results. Here, we describe in detail a rapid and accurate method for the measurement of peroxisomal pH, using the pHRed sensor protein and confocal microscopy of living mammalian cells. pHRed, a ratiometric sensor of pH, is targeted to the peroxisomes by virtue of a C-terminal targeting sequence. The probe has a maximum fluorescence emission at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm, allowing for ratiometric imaging and determination of intracellular pH in live cell microscopy.

Dynamic analysis of Lactobacillus delbrueckii subsp. bulgaricus CFL1 physiological characteristics during fermentation.

Science.gov (United States)

Rault, Aline; Bouix, Marielle; Béal, Catherine

2008-12-01

This study aimed at examining and comparing the relevance of various methods in order to discriminate different cellular states of Lactobacillus bulgaricus CFL1 and to improve knowledge on the dynamics of the cellular physiological state during growth and acidification. By using four fluorescent probes combined with multiparametric flow cytometry, membrane integrity, intracellular esterase activity, cellular vitality, membrane depolarization, and intracellular pH were quantified throughout fermentations. Results were compared and correlated with measurements of cultivability, acidification activity (Cinac system), and cellular ability to recover growth in fresh medium (Bioscreen system). The Cinac system and flow cytometry were relevant to distinguish different physiological states throughout growth. Lb. bulgaricus cells maintained their high viability, energetic state, membrane potential, and pH gradient in the late stationary phase, despite the gradual decrease of both cultivability and acidification activity. Viability and membrane integrity were maintained during acidification, at the expense of their cultivability and acidification activity. Finally, this study demonstrated that the physiological state during fermentation was strongly affected by intracellular pH and the pH gradient. The critical pHi of Lb. bulgaricus CFL1 was found to be equal to pH 5.8. Through linear relationships between dpH and cultivability and pHi and acidification activity, pHi and dpH well described the time course of metabolic activity, cultivability, and viability in a single analysis.

High Gradient Accelerator Research

International Nuclear Information System (INIS)

Temkin, Richard

2016-01-01

The goal of the MIT program of research on high gradient acceleration is the development of advanced acceleration concepts that lead to a practical and affordable next generation linear collider at the TeV energy level. Other applications, which are more near-term, include accelerators for materials processing; medicine; defense; mining; security; and inspection. The specific goals of the MIT program are: • Pioneering theoretical research on advanced structures for high gradient acceleration, including photonic structures and metamaterial structures; evaluation of the wakefields in these advanced structures • Experimental research to demonstrate the properties of advanced structures both in low-power microwave cold test and high-power, high-gradient test at megawatt power levels • Experimental research on microwave breakdown at high gradient including studies of breakdown phenomena induced by RF electric fields and RF magnetic fields; development of new diagnostics of the breakdown process • Theoretical research on the physics and engineering features of RF vacuum breakdown • Maintaining and improving the Haimson / MIT 17 GHz accelerator, the highest frequency operational accelerator in the world, a unique facility for accelerator research • Providing the Haimson / MIT 17 GHz accelerator facility as a facility for outside users • Active participation in the US DOE program of High Gradient Collaboration, including joint work with SLAC and with Los Alamos National Laboratory; participation of MIT students in research at the national laboratories • Training the next generation of Ph. D. students in the field of accelerator physics.

Response of Listeria monocytogenes to disinfection stress at the single-cell and population levels as monitored by intracellular pH measurements and viable-cell counts

DEFF Research Database (Denmark)

Kastbjerg, Vicky Gaedt; Nielsen, Dennis S.; Arneborg, Nils

2009-01-01

of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level...... by measuring intracellular pH (pHi) over time by fluorescence ratio imaging microscopy. pHi values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population...... was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pHi was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pHi 5 to 5.5) (P

Effects of potentially acidic air pollutants on the intracellular distribution and transport of plant growth regulators in mesophyll cells of leaves. Consequences on stress- and developmental physiology

Energy Technology Data Exchange (ETDEWEB)

Kremer, H.; Pfanz, H.; Hartung, W.

1987-07-11

The influence of SO/sub 2/ on the intracellular distribution of abscisic acid (ABA) and indole-acetic acid (IAA) in mesophyll cells of Picea abies, Tsuga americana and Hordeum vulgare was investigated. The compartmentation of ABA and IAA depends on intracellular pH-gradients. The hydrophilic anions ABA and IAA are accumulated in the alkaline cell compartments cytosol and chloroplasts, which act as anion traps for weak acids. Uptake of sulfur dioxide into leaves leads to an acidification of alkaline cell compartments, thus decreasing intracellular pH-gradients. Consequently this results in an increased release of plant growth regulators from the cell interior into the apoplast. Therefore the target cells of plant hormones i.e. meristems and stomates are exposed to altered hormone concentrations. Obviously this influences the regulation of cellular metabolism plant development and growth.

In Vivo Monitoring of pH, Redox Status, and Glutathione Using L-Band EPR for Assessment of Therapeutic Effectiveness in Solid Tumors

Science.gov (United States)

Bobko, Andrey A.; Eubank, Timothy D.; Voorhees, Jeffrey L.; Efimova, Olga V.; Kirilyuk, Igor A.; Petryakov, Sergey; Trofimiov, Dmitrii G.; Marsh, Clay B.; Zweier, Jay L.; Grigor’ev, Igor A.; Samouilov, Alexandre; Khramtsov, Valery V.

2011-01-01

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pHe), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pHe is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pHe by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pHe mapping was performed using recently proposed variable frequency proton–electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pHe and a difference of about 0.4 pH units between average pHe values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pHe, extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors. Magn Reson Med 000:000–000, 2011. PMID:22113626

Fabrication of triple-labeled polyelectrolyte microcapsules for localized ratiometric pH sensing.

Science.gov (United States)

Song, Xiaoxue; Li, Huanbin; Tong, Weijun; Gao, Changyou

2014-02-15

Encapsulation of pH sensitive fluorophores as reporting molecules provides a powerful approach to visualize the transportation of multilayer capsules. In this study, two pH sensitive dyes (fluorescein and oregon green) and one pH insensitive dye (rhodamine B) were simultaneously labeled on the microcapsules to fabricate ratiometric pH sensors. The fluorescence of the triple-labeled microcapsule sensors was robust and nearly independent of other intracellular species. With a dynamic pH measurement range of 3.3-6.5, the microcapsules can report their localized pH at a real time. Cell culture experiments showed that the microcapsules could be internalized by RAW 246.7 cells naturally and finally accumulated in acidic organelles with a pH value of 5.08 ± 0.59 (mean ± s.d.; n=162). Copyright © 2013 Elsevier Inc. All rights reserved.

Fluorescent ratiometric pH indicator SypHer2: applications in neuroscience and regenerative biology

Science.gov (United States)

Matlashov, Mikhail E.; Bogdanova, Yulia A.; Ermakova, Galina V.; Mishina, Natalia M.; Ermakova, Yulia G.; Nikitin, Evgeny S.; Balaban, Pavel M.; Okabe, Shigeo; Lukyanov, Sergey; Enikolopov, Grigori; Zaraisky, Andrey G.; Belousov, Vsevolod V.

2015-01-01

Background SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, a relatively low brightness of the indicator limits its use. Methods Here we designed a new version of pH-sensor - SypHer-2, that has up to three times brighter fluorescence signal in cultured mammalian cells compared to the SypHer. Results Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent temporal neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop which occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. Conclusions SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. General significance The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies. PMID:26259819

Fluorescent ratiometric pH indicator SypHer2: Applications in neuroscience and regenerative biology.

Science.gov (United States)

Matlashov, Mikhail E; Bogdanova, Yulia A; Ermakova, Galina V; Mishina, Natalia M; Ermakova, Yulia G; Nikitin, Evgeny S; Balaban, Pavel M; Okabe, Shigeo; Lukyanov, Sergey; Enikolopov, Grigori; Zaraisky, Andrey G; Belousov, Vsevolod V

2015-11-01

SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use. Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer. Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate. SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo. The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Ratiometric fluorescent sensing of pH values in living cells by dual-fluorophore-labeled i-motif nanoprobes.

Science.gov (United States)

Huang, Jin; Ying, Le; Yang, Xiaohai; Yang, Yanjing; Quan, Ke; Wang, He; Xie, Nuli; Ou, Min; Zhou, Qifeng; Wang, Kemin

2015-09-01

We designed a new ratiometric fluorescent nanoprobe for sensing pH values in living cells. Briefly, the nanoprobe consists of a gold nanoparticle (AuNP), short single-stranded oligonucleotides, and dual-fluorophore-labeled i-motif sequences. The short oligonucleotides are designed to bind with the i-motif sequences and immobilized on the AuNP surface via Au-S bond. At neutral pH, the dual fluorophores are separated, resulting in very low fluorescence resonance energy transfer (FRET) efficiency. At acidic pH, the i-motif strands fold into a quadruplex structure and leave the AuNP, bringing the dual fluorophores into close proximity, resulting in high FRET efficiency, which could be used as a signal for pH sensing. The nanoprobe possesses abilities of cellular transfection, enzymatic protection, fast response and quantitative pH detection. The in vitro and intracellular applications of the nanoprobe were demonstrated, which showed excellent response in the physiological pH range. Furthermore, our experimental results suggested that the nanoprobe showed excellent spatial and temporal resolution in living cells. We think that the ratiometric sensing strategy could potentially be applied to create a variety of new multicolor sensors for intracellular detection.

Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

Science.gov (United States)

Mundy, Dorothy I.; Li, Wei Ping; Luby-Phelps, Katherine; Anderson, Richard G. W.

2012-01-01

Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking. PMID:22238363

Highly Efficient Intracellular Protein Delivery by Cationic Polyethyleneimine-Modified Gelatin Nanoparticles

Directory of Open Access Journals (Sweden)

Ming-Ju Chou

2018-02-01

Full Text Available Intracellular protein delivery may provide a safe and non-genome integrated strategy for targeting abnormal or specific cells for applications in cell reprogramming therapy. Thus, highly efficient intracellular functional protein delivery would be beneficial for protein drug discovery. In this study, we generated a cationic polyethyleneimine (PEI-modified gelatin nanoparticle and evaluated its intracellular protein delivery ability in vitro and in vivo. The experimental results showed that the PEI-modified gelatin nanoparticle had a zeta potential of approximately +60 mV and the particle size was approximately 135 nm. The particle was stable at different biological pH values and temperatures and high protein loading efficiency was observed. The fluorescent image results revealed that large numbers of particles were taken up into the mammalian cells and escaped from the endosomes into the cytoplasm. In a mouse C26 cell-xenograft cancer model, particles accumulated in cancer cells. In conclusion, the PEI-modified gelatin particle may provide a biodegradable and highly efficient protein delivery system for use in regenerative medicine and cancer therapy.

Measurement of the Extracellular pH of Adherently Growing Mammalian Cells with High Spatial Resolution Using a Voltammetric pH Microsensor.

Science.gov (United States)

Munteanu, Raluca-Elena; Stǎnicǎ, Luciana; Gheorghiu, Mihaela; Gáspár, Szilveszter

2018-05-15

There are only a few tools suitable for measuring the extracellular pH of adherently growing mammalian cells with high spatial resolution, and none of them is widely used in laboratories around the world. Cell biologists very often limit themselves to measuring the intracellular pH with commercially available fluorescent probes. Therefore, we built a voltammetric pH microsensor and investigated its suitability for monitoring the extracellular pH of adherently growing mammalian cells. The voltammetric pH microsensor consisted of a 37 μm diameter carbon fiber microelectrode modified with reduced graphene oxide and syringaldazine. While graphene oxide was used to increase the electrochemically active surface area of our sensor, syringaldazine facilitated pH sensing through its pH-dependent electrochemical oxidation and reduction. The good sensitivity (60 ± 2.5 mV/pH unit), reproducibility (coefficient of variation ≤3% for the same pH measured with 5 different microsensors), and stability (pH drift around 0.05 units in 3 h) of the built voltammetric pH sensors were successfully used to investigate the acidification of the extracellular space of both cancer cells and normal cells. The results indicate that the developed pH microsensor and the perfected experimental protocol based on scanning electrochemical microscopy can reveal details of the pH regulation of cells not attainable with pH sensors lacking spatial resolution or which cannot be reproducibly positioned in the extracellular space.

Involvement of detergent-insoluble complexes in the intracellular transport of intestinal brush border enzymes

DEFF Research Database (Denmark)

Danielsen, E M

1995-01-01

A number of transmembrane digestive enzymes of the porcine small intestinal brush border membrane were found to be partially Triton X-100-insoluble at 0 degree C and colocalized in gradient centrifugation experiments with the GPI-anchored alkaline phosphatase in low-density, detergent-insoluble c...... intracellularly. I therefore propose that, in the enterocyte, the brush border enzymes are targeted directly from the trans-Golgi network toward the apical cell surface......., and their insolubility increased to that of the steady-state level soon after they achieved their mature, complex glycosylation, i.e., after passage through the Golgi complex. Detergent-insoluble complexes isolated by density gradient centrifugation were highly enriched in brush border enzymes, and the enrichment...

Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

Science.gov (United States)

García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

2013-01-01

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

Cadmium uptake in Elodea canadensis leaves and its interference with extra- and intra-cellular pH.

Science.gov (United States)

Javed, M T; Lindberg, S; Greger, M

2014-05-01

This study investigated cadmium (Cd) uptake in Elodea canadensis shoots under different photosynthetic conditions, and its effects on internal (cytosolic) and external pH. The plants were grown under photosynthetic (light) or non-photosynthetic (dark or in the presence of a photosynthetic inhibitor) conditions in the presence or absence of CdCl2 (0.5 μm) in a medium with a starting pH of 5.0. The pH-sensitive dye BCECF-AM was used to monitor cytosolic pH changes in the leaves. Cadmium uptake in protoplasts and leaves was detected with a Cd-specific fluorescent dye, Leadmium Green AM, and with atomic absorption spectrophotometry. During cultivation for 3 days without Cd, shoots of E. canadensis increased the pH of the surrounding water, irrespective of the photosynthetic conditions. This medium alkalisation was higher in the presence of CdCl2 . Moreover, the presence of Cd also increased the cation exchange capacity of the shoots. The total Cd uptake by E. canadensis shoots was independent of photosynthetic conditions. Protoplasts from plants exposed to 0.5 μm CdCl2 for 3 days did not exhibit significant change in cytosolic [Cd(2+)] or pH. However, exposure to CdCl2 for 7 days resulted in increased cytosolic [Cd(2+) ] as well as pH. The results suggest that E. canadensis subjected to a low CdCl2 concentration initially sequesters Cd into the apoplasm, but under prolonged exposure, Cd is transported into the cytosol and subsequently alters cytosolic pH. In contrast, addition of 10-50 μm CdCl2 directly to protoplasts resulted in immediate uptake of Cd into the cytosol. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system

OpenAIRE

Raimondo, Joseph V.; Joyce, Bradley; Kay, Louise; Schlagheck, Theresa; Newey, Sarah E.; Srinivas, Shankar; Akerman, Colin J.

2013-01-01

Within the nervous system, intracellular Cl− and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl− and pH are often co-regulated, and network activity results in the movement of both Cl− and H+. Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl− and pH sensors have been described previously, these either l...

Ingenious pH-sensitive dextran/mesoporous silica nanoparticles based drug delivery systems for controlled intracellular drug release.

Science.gov (United States)

Zhang, Min; Liu, Jia; Kuang, Ying; Li, Qilin; Zheng, Di-Wei; Song, Qiongfang; Chen, Hui; Chen, Xueqin; Xu, Yanglin; Li, Cao; Jiang, Bingbing

2017-05-01

In this work, dextran, a polysaccharide with excellent biocompatibility, is applied as the "gatekeeper" to fabricate the pH-sensitive dextran/mesoporous silica nanoparticles (MSNs) based drug delivery systems for controlled intracellular drug release. Dextran encapsulating on the surface of MSNs is oxidized by NaIO 4 to obtain three kinds of dextran dialdehydes (PADs), which are then coupled with MSNs via pH-sensitive hydrazone bond to fabricate three kinds of drug carriers. At pH 7.4, PADs block the pores to prevent premature release of anti-cancer drug doxorubicin hydrochloride (DOX). However, in the weakly acidic intracellular environment (pH∼5.5) the hydrazone can be ruptured; and the drug can be released from the carriers. The drug loading capacity, entrapment efficiency and release rates of the drug carriers can be adjusted by the amount of NaIO 4 applied in the oxidation reaction. And from which DOX@MSN-NH-N=C-PAD 10 is chosen as the most satisfactory one for the further in vitro cytotoxicity studies and cellular uptake studies. The results demonstrate that DOX@MSN-NH-N=C-PAD 10 with an excellent pH-sensitivity can enter HeLa cells to release DOX intracellular due to the weakly acidic pH intracellular and kill the cells. In our opinion, the ingenious pH-sensitive drug delivery systems have application potentials for cancer therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Intracellular pH in Gastric and Rectal Tissue Post Cardiac Arrest

Science.gov (United States)

Fisher, Elaine M.; Steiner, Richard P.; LaManna, Joseph C.

We directly measured pHi using the pH sensitive dye, neutral red. We defined pHi for rectal and gastric tissue in whole tissue and by layer under control and arrest conditions. Fifteen minutes of arrest was not sufficient time to alter the pHi at the rectal or gastric site. On initial inspection, the stomach may be more sensitive to ischemic changes than the rectum. Understanding the mechanism by which PCO2 generation is used to track clinical changes is vital to the early detection of tissue dysoxia in order to effectively treat and manage critically ill patients.

pH-Sensitive nanoparticles as smart carriers for selective intracellular drug delivery to tumor.

Science.gov (United States)

Li, Xin-Xin; Chen, Jing; Shen, Jian-Min; Zhuang, Ran; Zhang, Shi-Qi; Zhu, Zi-Yun; Ma, Jing-Bo

2018-05-05

Herein, a smart pH-sensitive nanoparticle (DGL-PEG-Tat-KK-DMA-DOX) was prepared to achieve the selective intracellular drug delivery. In this nanoparticle, a PEG-grafted cell penetrating peptide (PEG-Tat-KK) was designed and acted as the cell penetrating segment. By introducing the pH-sensitive amide bonds between the peptide and blocking agent (2,3-dimethylmaleic anhydride, DMA), the controllable moiety (PEG-Tat-KK-DMA) endowed the nanoparticle with a charge-switchable shell and temporarily blocked penetrating function, thus improving the specific internalization. Besides, dendrigraft poly-L-lysine (DGL) used as the skeleton can greatly improve the drug loading because of the highly dendritic framework. Under the stimuli of acidic pH, this nanoparticle exhibited a remarkable charge-switchable property. The drug release showed an expected behavior with little release in the neutral pH media but relatively fast release in the acidic media. The in vitro experiments revealed that the cellular uptake and cytotoxicity were significantly enhanced after the pH was decreased. In vivo biodistribution and antitumor research indicated that the nanoparticle had noteworthy specificity and antitumor efficacy with a tumor inhibition rate of 79.7%. These results verified this nanoparticle could efficiently improve the selective intracellular delivery and possessed a great potential in tumor treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

[Ph-Sensor Properties of a Fluorescent Protein from Dendronephthya sp].

Science.gov (United States)

Pakhomov, A A; Chertkova, R V; Martynov, V I

2015-01-01

Genetically encoded biosensors based on fluorescent proteins are now widely applicable for monitoring pH changes in live cells. Here, we have shown that a fluorescent protein from Dendronephthya sp. (DendFP) exhibits a pronounced pH-sensitivity. Unlike most of known genetically encoded pH-sensors, fluorescence of the protein is not quenched upon medium acidification, but is shifting from the red to green spectral range. Therefore, quantitative measurements of intracellular pH are feasible by ratiometric comparison of emission intensities in the red and green spectral ranges, which makes DendFP advantageous compared with other genetically encoded pH-sensors.

Intracellular product recycling in high succinic acid producing yeast at low pH

NARCIS (Netherlands)

Wahl, S.A.; Bernal Martinez, C.; Zhao, Zheng; van Gulik, W.M.; Jansen, Mickel L.A.

2017-01-01

Background: The metabolic engineering of Saccharomyces cerevisiae for the production of succinic acid has progressed dramatically, and a series of high-producing hosts are available. At low cultivation pH and high titers, the product transport can become bidirectional, i.e. the acid is reentering

Bicarbonate/chloride antiport in Vero cells: II. Mechanisms for bicarbonate-dependent regulation of intracellular pH

International Nuclear Information System (INIS)

Olsnes, S.; Ludt, J.; Tonnessen, T.I.; Sandvig, K.

1987-01-01

The rates of bicarbonate-dependent uptake and efflux of 22 Na + in Vero cells were studied and compared with the uptake and efflux of 36 Cl - . Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten-fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na + was much less affected by changes in pH. The bicarbonate-linked uptake of 22 Na + was dependent on internal Cl- but not on internal Na + . At a constant external concentration of HCO 3 -, the amount of 22 Na + associated with the cells increased when the internal concentration of HCO 3 - decreased and vice versa, which is compatible with the possibility that the ion pair NaCO 3 - is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36 Cl - both in the absence and presence of Na + . At alkaline internal pH, HCO 3 - stimulated the efflux of 36 Cl - from preloaded cells, while at acidic internal pH both Na + and HCO 3 - were required to induce 36 Cl - efflux. We propose a model for how bicarbonate-dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH(-)-equivalents in opposite directions across the plasma membrane

Tris-hydroxymethyl-aminomethane enhances capsaicin-induced intracellular Ca2+ influx through transient receptor potential V1 (TRPV1 channels

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Satoshi Murakami

2016-02-01

Full Text Available Non-selective transient receptor potential vanilloid (TRPV cation channels are activated by various insults, including exposure to heat, acidity, and the compound capsaicin, resulting in sensations of pain in the skin, visceral organs, and oral cavity. Recently, TRPV1 activation was also demonstrated in response to basic pH elicited by ammonia and intracellular alkalization. Tris-hydroxymethyl aminomethane (THAM is widely used as an alkalizing agent; however, the effects of THAM on TRPV1 channels have not been defined. In this study, we characterized the effects of THAM-induced TRPV1 channel activation in baby hamster kidney cells expressing human TRPV1 (hTRPV1 and the Ca2+-sensitive fluorescent sensor GCaMP2 by real-time confocal microscopy. Notably, both capsaicin (1 μM and pH 6.5 buffer elicited steep increases in the intracellular Ca2+ concentration ([Ca2+]i, while treatment with THAM (pH 8.5 alone had no effect. However, treatment with THAM (pH 8.5 following capsaicin application elicited a profound, long-lasting increase in [Ca2+]i that was completely inhibited by the TRPV1 antagonist capsazepine. Taken together, these results suggest that hTRPV1 pre-activation is required to provoke enhanced, THAM-induced [Ca2+]i increases, which could be a mechanism underlying pain induced by basic pH.

The Influence of Virus Infection on the Extracellular pH of the Host Cell Detected on Cell Membrane.

Science.gov (United States)

Liu, Hengjun; Maruyama, Hisataka; Masuda, Taisuke; Honda, Ayae; Arai, Fumihito

2016-01-01

Influenza virus infection can result in changes in the cellular ion levels at 2-3 h post-infection. More H(+) is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H(+) during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H(+) from the intracellular compartment. Increased H(+) export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (ϕ1 μm) containing Rhodamine B and Fluorescein isothiocyanate (FITC). The fluorescence intensity of FITC can respond to both pH and temperature. So Rhodamine B was also introduced in the sensor for temperature compensation. Then the pH can be measured after temperature compensation. The sensor was adhered to cell membrane for extracellular pH measurement. The results showed that the multiplication of influenza virus in host cell decreased extracellular pH of the host cell by 0.5-0.6 in 4 h after the virus bound to the cell membrane, compared to that in uninfected cells. Immunostaining revealed the presence of viral PB1 protein in the nucleus of virus-bound cells that exhibited extracellular pH changes, but no PB1 protein are detected in virus-unbound cells where the extracellular pH remained constant.

The influence of virus infection on the extracellular pH of the host cell detected on cell membrane

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Hengjun Liu

2016-08-01

Full Text Available Influenza virus infection can result in changes in the cellular ion levels at 2–3 hours post-infection. More H+ is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H+ during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H+ from the intracellular compartment. Increased H+ export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (1μm containing Rhodamine B and Fluorescein isothiocyanate (FITC. The fluorescence intensity of FITC can respond to both pH and temperature. So Rhodamine B was also introduced in the sensor for temperature compensation. Then the pH can be measured after temperature compensation. The sensor was adhered to cell membrane for extracellular pH measurement. The results showed that the multiplication of influenza virus in host cell decreased extracellular pH of the host cell by 0.5–0.6 in 4 hours after the virus bound to the cell membrane, compared to that in uninfected cells. Immunostaining revealed the presence of viral PB1 subunits in the nucleus of virus-bound cells that exhibited extracellular pH changes, but no PB1 subunits are detected in virus-unbound cells where the extracellular pH remained constant.

Contrasting trends in distribution of four major planktonic betaproteobacterial groups along a pH gradient of epilimnia of 72 freshwater habitats.

Science.gov (United States)

Jezbera, Jan; Jezberová, Jitka; Koll, Ulrike; Horňák, Karel; Šimek, Karel; Hahn, Martin W

2012-08-01

The distribution and abundance of Betaproteobacteria and three of its genera - Limnohabitans (R-BT065 lineage), Polynucleobacter (including subclusters Polynucleobacter necessarius and Polynucleobacter acidiphobus/Polynucleobacter difficilis), and Methylophilus - across the epilimnia of 72 limnologically diverse freshwater habitats were investigated using fluorescence in situ hybridization. Moreover, seasonal development of Betaproteobacteria subgroups along the longitudinal axis of a reservoir was followed. Betaproteobacteria comprised on average 29.1%, Polynucleobacter 11.6%, P. necessarius 10.1%, P. acidiphobus/difficilis 0.5%, Limnohabitans 8.9%, and Methylophilus 0.9% of total bacterioplankton cells in the investigated habitats. Polynucleobacter necessarius and Limnohabitans coexisted in the majority of habitats but showed contrasting abundance patterns along the pH gradient of habitats (pH, 3.8-8.5). The observed distribution patterns could theoretically be explained by different preferences for substrate sources, that is, substances of humic origin in acidic waters and algal-derived substances in alkaline waters. However, substrate utilization patterns observed in laboratory experiments indicate no coherent group-specific differences in substrate preferences. Interestingly, similar distribution patterns were revealed for Limnohabitans and P. acidiphobus/difficilis, suggesting similar ecological adaptations of these distantly related taxa. Our findings further emphasize that at least two taxa of freshwater Betaproteobacteria represent ecologically diversified groups. Investigations at higher phylogenetic resolution are required for obtaining further insights into their ecology. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.

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Zhdanov, A V; Golubeva, A V; Okkelman, I A; Cryan, J F; Papkovsky, D B

2015-10-01

O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-μm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 μM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O2 gradients of up to 40 μM across the cell, or ∼0.6 μM/μm. Deeply deoxygenated (5-15 μM O2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O2 gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O2 distribution potentially can be used for the modeling of intracellular O2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O2-dependent (patho)physiological processes in the bladder and other tissues. Copyright © 2015 the American Physiological Society.

The electrochemical H+ gradient in the yeast Rhodotorula glutinis

NARCIS (Netherlands)

Höfer, M.H.; Nicolaij, K.; Robillard, G.T.

1985-01-01

The electrochemical gradient of protons, delta mu H+, was estimated in the obligatory aerobic yeast Rhodotorula glutinis in the pH0 range from 3 to 8.5. The membrane potential, delta psi, was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP+) for negative

Live tissue imaging shows reef corals elevate pH under their calcifying tissue relative to seawater.

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Alexander Venn

Full Text Available The threat posed to coral reefs by changes in seawater pH and carbonate chemistry (ocean acidification raises the need for a better mechanistic understanding of physiological processes linked to coral calcification. Current models of coral calcification argue that corals elevate extracellular pH under their calcifying tissue relative to seawater to promote skeleton formation, but pH measurements taken from the calcifying tissue of living, intact corals have not been achieved to date. We performed live tissue imaging of the reef coral Stylophora pistillata to determine extracellular pH under the calcifying tissue and intracellular pH in calicoblastic cells. We worked with actively calcifying corals under flowing seawater and show that extracellular pH (pHe under the calicoblastic epithelium is elevated by ∼0.5 and ∼0.2 pH units relative to the surrounding seawater in light and dark conditions respectively. By contrast, the intracellular pH (pHi of the calicoblastic epithelium remains stable in the light and dark. Estimates of aragonite saturation states derived from our data indicate the elevation in subcalicoblastic pHe favour calcification and may thus be a critical step in the calcification process. However, the observed close association of the calicoblastic epithelium with the underlying crystals suggests that the calicoblastic cells influence the growth of the coral skeleton by other processes in addition to pHe modification. The procedure used in the current study provides a novel, tangible approach for future investigations into these processes and the impact of environmental change on the cellular mechanisms underpinning coral calcification.

Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

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Marion Le Coadic

Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

pH regulation in sensitive and multidrug resistant Ehrlich ascites tumor cells

DEFF Research Database (Denmark)

Litman, Thomas; Pedersen, S F; Kramhøft, B

1998-01-01

Maintenance and regulation of intracellular pH (pHi) was studied in wild-type Ehrlich ascites tumor cells (EHR2) and five progressively daunorubicin-resistant, P-glycoprotein (P-gp)-expressing strains, the maximally resistant of which is EHR2/1.3. Steady-state pHi was similar in cells expressing...

pH sensing and regulation in cancer.

Science.gov (United States)

Damaghi, Mehdi; Wojtkowiak, Jonathan W; Gillies, Robert J

2013-12-17

Cells maintain intracellular pH (pHi) within a narrow range (7.1-7.2) by controlling membrane proton pumps and transporters whose activity is set by intra-cytoplasmic pH sensors. These sensors have the ability to recognize and induce cellular responses to maintain the pHi, often at the expense of acidifying the extracellular pH. In turn, extracellular acidification impacts cells via specific acid-sensing ion channels (ASICs) and proton-sensing G-protein coupled receptors (GPCRs). In this review, we will discuss some of the major players in proton sensing at the plasma membrane and their downstream consequences in cancer cells and how these pH-mediated changes affect processes such as migration and metastasis. The complex mechanisms by which they transduce acid pH signals to the cytoplasm and nucleus are not well understood. However, there is evidence that expression of proton-sensing GPCRs such as GPR4, TDAG8, and OGR1 can regulate aspects of tumorigenesis and invasion, including cofilin and talin regulated actin (de-)polymerization. Major mechanisms for maintenance of pHi homeostasis include monocarboxylate, bicarbonate, and proton transporters. Notably, there is little evidence suggesting a link between their activities and those of the extracellular H(+)-sensors, suggesting a mechanistic disconnect between intra- and extracellular pH. Understanding the mechanisms of pH sensing and regulation may lead to novel and informed therapeutic strategies that can target acidosis, a common physical hallmark of solid tumors.

OptiPrep? Density Gradient Solutions for Macromolecules and Macromolecular Complexes

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John Graham

2002-01-01

Full Text Available Any density gradient for the isolation of mammalian cells should ideally only expose the sedimenting particles to an increasing concentration of the gradient solute. Thus they will experience only an increasing density and viscosity, other parameters such as osmolality, pH, ionic strength and the concentration of important additives (such as EDTA or divalent cations should remain as close to constant as possible. This Protocol Article describes the strategies for the dilution of OptiPrep™ in order to prepare such solutions for mammalian cells.

Miniaturized pH Sensors Based on Zinc Oxide Nanotubes/Nanorods

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Magnus Willander

2009-11-01

Full Text Available ZnO nanotubes and nanorods grown on gold thin film were used to create pH sensor devices. The developed ZnO nanotube and nanorod pH sensors display good reproducibility, repeatability and long-term stability and exhibit a pH-dependent electrochemical potential difference versus an Ag/AgCl reference electrode over a large dynamic pH range. We found the ZnO nanotubes provide sensitivity as high as twice that of the ZnO nanorods, which can be ascribed to the fact that small dimensional ZnO nanotubes have a higher level of surface and subsurface oxygen vacancies and provide a larger effective surface area with higher surface-to-volume ratio as compared to ZnO nanorods, thus affording the ZnO nanotube pH sensor a higher sensitivity. Experimental results indicate ZnO nanotubes can be used in pH sensor applications with improved performance. Moreover, the ZnO nanotube arrays may find potential application as a novel material for measurements of intracellular biochemical species within single living cells.

Lateral shearing optical gradient force in coupled nanobeam photonic crystal cavities

Energy Technology Data Exchange (ETDEWEB)

Du, Han; Zhang, Xingwang; Chau, Fook Siong; Zhou, Guangya, E-mail: mpezgy@nus.edu.sg [Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117575 (Singapore); Deng, Jie [Institute of Materials Research and Engineering, 2 Fusionopolis Way, Innovis, #08-03, Singapore 138634 (Singapore); Zhao, Yunshan [Department of Electrical and Computer Engineering, National University of Singapore, 4 Engineering Drive 3, Singapore 117583 (Singapore)

2016-04-25

We report the experimental observation of lateral shearing optical gradient forces in nanoelectromechanical systems (NEMS) controlled dual-coupled photonic crystal (PhC) nanobeam cavities. With an on-chip integrated NEMS actuator, the coupled cavities can be mechanically reconfigured in the lateral direction while maintaining a constant coupling gap. Shearing optical gradient forces are generated when the two cavity centers are laterally displaced. In our experiments, positive and negative lateral shearing optical forces of 0.42 nN and 0.29 nN are observed with different pumping modes. This study may broaden the potential applications of the optical gradient force in nanophotonic devices and benefit the future nanooptoelectromechanical systems.

pH imaging of mouse kidneys in vivo using a frequency-dependent paraCEST agent

Science.gov (United States)

Wu, Yunkou; Zhang, Shanrong; Soesbe, Todd C.; Yu, Jing; Vinogradov, Elena; Lenkinski, Robert E.; Sherry, A. Dean

2015-01-01

Purpose This study explored the feasibility of using a pH responsive paraCEST agent to image the pH gradient in kidneys of healthy mice. Methods CEST signals were acquired on an Agilent 9.4 T small animal MRI system using a steady-state gradient echo pulse sequence after a bolus injection of agent. The magnetic field inhomogeneity across each kidney was corrected using the WASSR method and pH maps were calculated by measuring the frequency of water exchange signal arising from the agent. Results Dynamic CEST studies demonstrated that the agent was readily detectable in kidneys only between 4 to 12 min post-injection. The CEST images showed a higher signal intensity in the pelvis and calyx regions and lower signal intensity in the medulla and cortex regions. The pH maps reflected tissue pH values spanning from 6.0 to 7.5 in kidneys of healthy mice. Conclusion This study demonstrated that pH maps of the kidney can be imaged in vivo by measuring the pH-dependent chemical shift of a single water exchange CEST peak without prior knowledge of the agent concentration in vivo. The results demonstrate the potential of using a simple frequency-dependent paraCEST agent for mapping tissue pH in vivo. PMID:26173637

Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

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Markham Richard B

2005-09-01

Full Text Available Abstract Background Both cell-associated and cell-free HIV virions are present in semen and cervical secretions of HIV-infected individuals. Thus, topical microbicides may need to inactivate both cell-associated and cell-free HIV to prevent sexual transmission of HIV/AIDS. To determine if the mild acidity of the healthy vagina and acid buffering microbicides would prevent transmission by HIV-infected leukocytes, we measured the effect of pH on leukocyte motility, viability and intracellular pH and tested the ability of an acidic buffering microbicide (BufferGel® to prevent the transmission of cell-associated HIV in a HuPBL-SCID mouse model. Methods Human lymphocyte, monocyte, and macrophage motilities were measured as a function of time and pH using various acidifying agents. Lymphocyte and macrophage motilities were measured using video microscopy. Monocyte motility was measured using video microscopy and chemotactic chambers. Peripheral blood mononuclear cell (PBMC viability and intracellular pH were determined as a function of time and pH using fluorescent dyes. HuPBL-SCID mice were pretreated with BufferGel, saline, or a control gel and challenged with HIV-1-infected human PBMCs. Results Progressive motility was completely abolished in all cell types between pH 5.5 and 6.0. Concomitantly, at and below pH 5.5, the intracellular pH of PBMCs dropped precipitously to match the extracellular medium and did not recover. After acidification with hydrochloric acid to pH 4.5 for 60 min, although completely immotile, 58% of PBMCs excluded ethidium homodimer-1 (dead-cell dye. In contrast, when acidified to this pH with BufferGel, a microbicide designed to maintain vaginal acidity in the presence of semen, only 4% excluded dye at 10 min and none excluded dye after 30 min. BufferGel significantly reduced transmission of HIV-1 in HuPBL-SCID mice (1 of 12 infected compared to saline (12 of 12 infected and a control gel (5 of 7 infected. Conclusion These

Effects of high-gradient magnetic fields on living cell machinery

International Nuclear Information System (INIS)

Zablotskii, V; Lunov, O; Kubinova, S; Polyakova, T; Dejneka, A; Sykova, E

2016-01-01

A general interest in biomagnetic effects is related to fundamental studies of the influence of magnetic fields on living objects on the cellular and whole organism levels. Emerging technologies offer new directions for the use of high-gradient magnetic fields to control cell machinery and to understand the intracellular biological processes of the emerging field of nanomedicine. In this review we aim at highlighting recent advances made in identifying fundamental mechanisms by which magnetic gradient forces act on cell fate specification and cell differentiation. The review also provides an analysis of the currently available magnetic systems capable of generating magnetic fields with spatial gradients of up to 10 MT m −1 , with the focus on their suitability for use in cell therapy. Relationships between experimental factors and underlying biophysical mechanisms and assumptions that would ultimately lead to a deeper understanding of cell machinery and the development of more predictive models for the evaluation of the effects of magnetic fields on cells, tissue and organisms are comprehensively discussed. (topical review)

Settlement pattern of Posidonia oceanica epibionts along a gradient of ocean acidification: an approach with mimics

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L. DONNARUMMA

2014-02-01

Full Text Available Effects of ocean acidification (OA on the colonization/settlement pattern of the epibiont community of the leaves and rhizomesof the Mediterranean seagrass,Posidoniaoceanica, have been studied at volcanic CO2vents off Ischia (Italy, using “mimics”as artificial substrates. The experiments were conducted in shallowPosidoniastands (2-3 m depth, in three stations on the northand three on the south sides of the study area, distributed along a pH gradient. At each station, 4 rhizome mimics and 6 artificialleaves were collected every three months (Sept 2009-Sept 2010. The epibionts on both leaf and rhizome mimics showed clearchanges along the pH gradient; coralline algae and calcareous invertebrates (bryozoans, serpulid polychaetes and barnacles weredominant at control stations but progressively disappeared at the most acidified stations. In these extremely low pH sites theassemblage was dominated by filamentous algae and non calcareous taxa such as hydroids and tunicates. Settlement pattern onthe artificial leaves and rhizome mimics over time showed a consistent distribution pattern along the pH gradient and highlightedthe peak of recruitment of the various organisms in different periods according to their life history.Posidoniamimics at theacidified station showed a poor and very simplified assemblage where calcifying epibionts seemed less competitive for space. Thisprofound difference in epiphyte communities in low pH conditions suggests cascading effects on the food web of the meadow and,consequently, on the functioning of the system

A dual pH and temperature responsive polymeric fluorescent sensor and its imaging application in living cells.

Science.gov (United States)

Yin, Liyan; He, Chunsheng; Huang, Chusen; Zhu, Weiping; Wang, Xin; Xu, Yufang; Qian, Xuhong

2012-05-11

A polymeric fluorescent sensor PNME, consisting of A4 and N-isopropylacrylamide (NIPAM) units, was synthesized. PNME exhibited dual responses to pH and temperature, and could be used as an intracellular pH sensor for lysosomes imaging. Moreover, it also could sense different temperature change in living cells at 25 and 37 °C, respectively. This journal is © The Royal Society of Chemistry 2012

Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability.

Science.gov (United States)

Suffrian, K; Schulz, K G; Gutowska, M A; Riebesell, U; Bleich, M

2011-05-01

• To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. • Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pH(i)). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H(+), CO(2) and HCO(3)(-). • Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pH(i) unit. pH(i) acutely followed the pH of seawater (pH(e)) in a linear fashion between pH(e) values of 6.5 and 9 with a slope of 0.44 per pH(e) unit. pH(i) was nearly insensitive to changes in seawater CO(2) at constant pH(e) and HCO(3)(-). An increase in extracellular HCO(3)(-) resulted in a slight intracellular acidification. In the presence of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pH(e) on pH(i). • The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pH(i) homeostasis involves a DIDS-sensitive mechanism. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

pH and the cytotoxicity of fluoride in an animal cell culture system

International Nuclear Information System (INIS)

Helgeland, K.; Leirskar, J.

1976-01-01

To investigate the mechanism for the toxicity of silicate cement as observed in a cell culture system, the effects of pH and fluoride were tested on human epithelial cells (NCTC 2544). At pH 7.3, fluoride concentrations from 15 to 25 μg/ml (0.79 to 1.3 mM) had a growth inhibitory effect. When the pH of the incubation medium was lowered to the range 7.0 to 6.4, an enhanced cytotoxic effect of fluoride was found, and even at 5 to 10 μg/ml growth inhibition occurred. Concomitant with the enhanced cytotoxicity of fluoride at low pH, there was an increased utilization of glucose and formation of lactate. Upon lowering the pH of the incubation medium from 7.4 to 6.7, a twofold increase in the intracellular concentration of fluoride was found. (author)

The Semen pH Affects Sperm Motility and Capacitation.

Science.gov (United States)

Zhou, Ji; Chen, Li; Li, Jie; Li, Hongjun; Hong, Zhiwei; Xie, Min; Chen, Shengrong; Yao, Bing

2015-01-01

As the chemical environment of semen can have a profound effect on sperm quality, we examined the effect of pH on the motility, viability and capacitation of human sperm. The sperm in this study was collected from healthy males to avoid interference from other factors. The spermatozoa cultured in sperm nutrition solution at pH 5.2, 6.2, 7.2 and 8.2 were analyzed for sperm total motility, progressive motility (PR), hypo-osmotic swelling (HOS) rate, and sperm penetration. Our results showed that these parameters were similar in pH 7.2 and 8.2 sperm nutrition solutions, but decreased in pH 5.2 and 6.2 solutions. The HOS rate exhibited positive correlation with the sperm total motility and PR. In addition, the sperm Na(+)/K(+)-ATPase activity at different pHs was measured, and the enzyme activity was significantly lower in pH 5.2 and 6.2 media, comparing with that in pH 8.2 and pH 7.2 solutions. Using flow cytometry (FCM) and laser confocal scanning microscopy (LCSM) analysis, the intracellular Ca2(+ )concentrations of sperm cultured in sperm capacitation solution at pH 5.2, 6.2, 7.2 and 8.2 were determined. Compared with that at pH 7.2, the mean fluorescence intensity of sperm in pH 5.2 and 6.2 media decreased significantly, while that of pH 8.2 group showed no difference. Our results suggested that the declined Na(+)/K(+)-ATPase activity at acidic pHs result in decreased sperm movement and capacitation, which could be one of the mechanisms of male infertility.

Self-assembled nanocomplexes of anionic pullulan and polyallylamine for DNA and pH-sensitive intracellular drug delivery

Science.gov (United States)

Vora, Lalit; Tyagi, Monica; Patel, Ketan; Gupta, Sanjay; Vavia, Pradeep

2014-12-01

The amalgamation of chemotherapy and gene therapy is promising treatment option for cancer. In this study, novel biocompatible self-assembled nanocomplexes (NCs) between carboxylmethylated pullulan t335 (CMP) with polyallylamine (CMP-PAA NCs) were developed for plasmid DNA (pDNA) and pH-sensitive doxorubicin (DOX) delivery. DOX was conjugated to CMP (DOX-CMP) via hydrazone and confirmed by FTIR and 1H-NMR. In vitro release studies of pH-sensitive DOX-CMP conjugate showed 23 and 85 % release after 48 h at pH 7.4 (physiological pH) and pH 5 (intracellular/tumoral pH), respectively. The CMP-PAA NCs or DOX-CMP-PAA NCs self-assembled into a nanosized (successful cellular uptake of DOX-CMP-PAA NCs in HEK293 cells. Thus, NCs hold great potential for targeted pDNA and pH-sensitive intratumoral drug delivery.

Polymeric nanoparticles for the intracellular delivery of paclitaxel in lung and breast cancer

Science.gov (United States)

Zubris, Kimberly Ann Veronica

Nanoparticles are useful for addressing many of the difficulties encountered when administering therapeutic compounds. Nanoparticles are able to increase the solubility of hydrophobic drugs, improve pharmacokinetics through sustained release, alter biodistribution, protect sensitive drugs from low pH environments or enzymatic alteration, and, in some cases, provide targeting of the drug to the desired tissues. The use of functional nanocarriers can also provide controlled intracellular delivery of a drug. To this end, we have developed functional pH-responsive expansile nanoparticles for the intracellular delivery of paclitaxel. The pH-responsiveness of these nanoparticles occurs due to a hydrophobic to hydrophilic transition of the polymer occurring under mildly acidic conditions. These polymeric nanoparticles were systematically evaluated for the delivery of paclitaxel in vitro and in vivo to improve local therapy for lung and breast cancers. Nanoparticles were synthesized using a miniemulsion polymerization process and were subsequently characterized and found to swell when exposed to acidic environments. Paclitaxel was successfully encapsulated within the nanoparticles, and the particles exhibited drug release at pH 5 but not at pH 7.4. In addition, the uptake of nanoparticles was observed using flow cytometry, and the anticancer efficacy of the paclitaxel-loaded nanoparticles was measured using cancer cell lines in vitro. The potency of the paclitaxel-loaded nanoparticles was close to that of free drug, demonstrating that the drug was effectively delivered by the particles and that the particles could act as an intracellular drug depot. Following in vitro characterization, murine in vivo studies demonstrated the ability of the paclitaxel-loaded responsive nanoparticles to delay recurrence of lung cancer and to prevent establishment of breast cancer in the mammary fat pads with higher efficacy than paclitaxel alone. In addition, the ability of nanoparticles to

Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling

Science.gov (United States)

Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; White, Thomas W.; Brink, Peter R.

2011-01-01

We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na+ that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ∼330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens’ circulation of Na+ was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium. PMID:21624945

Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties.

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Chafik, Abdelbasset; Essamadi, Abdelkhalid; Çelik, Safinur Yildirim; Mavi, Ahmet

2017-12-01

Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al 3+ , Cd 2+ and Mg 2+ , whereas Ca 2+ , Co 2+ and Ni 2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The K m and V max of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with K i value of 14.43μM, the IC 50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of a molecular pH sensor in coral.

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Barott, Katie L; Barron, Megan E; Tresguerres, Martin

2017-11-15

Maintaining stable intracellular pH (pHi) is essential for homeostasis, and requires the ability to both sense pH changes that may result from internal and external sources, and to regulate downstream compensatory pH pathways. Here we identified the cAMP-producing enzyme soluble adenylyl cyclase (sAC) as the first molecular pH sensor in corals. sAC protein was detected throughout coral tissues, including those involved in symbiosis and calcification. Application of a sAC-specific inhibitor caused significant and reversible pHi acidosis in isolated coral cells under both dark and light conditions, indicating sAC is essential for sensing and regulating pHi perturbations caused by respiration and photosynthesis. Furthermore, pHi regulation during external acidification was also dependent on sAC activity. Thus, sAC is a sensor and regulator of pH disturbances from both metabolic and external origin in corals. Since sAC is present in all coral cell types, and the cAMP pathway can regulate virtually every aspect of cell physiology through post-translational modifications of proteins, sAC is likely to trigger multiple homeostatic mechanisms in response to pH disturbances. This is also the first evidence that sAC modulates pHi in any non-mammalian animal. Since corals are basal metazoans, our results indicate this function is evolutionarily conserved across animals. © 2017 The Author(s).

Protein Alterations in Infiltrating Ductal Carcinomas of the Breast as Detected by Nonequilibrium pH Gradient Electrophoresis and Mass Spectrometry

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Maria Kabbage

2008-01-01

Full Text Available Improvement of breast-cancer detection through the identification of potential cancer biomarkers is considered as a promising strategy for effective assessment of the disease. The current study has used nonequilibrium pH gradient electrophoresis with subsequent analysis by mass spectrometry to identify protein alterations in invasive ductal carcinomas of the breast from Tunisian women. We have identified multiple protein alterations in tumor tissues that were picked, processed, and unambiguously assigned identities by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF. The proteins identified span a wide range of functions and are believed to have potential clinical applications as cancer biomarkers. They include glycolytic enzymes, molecular chaperones, cytoskeletal-related proteins, antioxydant enzymes, and immunologic related proteins. Among these proteins, enolase 1, phosphoglycerate kinase 1, deoxyhemoglobin, Mn-superoxyde dismutase, α-B-crystallin, HSP27, Raf kinase inhibitor protein, heterogeneous nuclear ribonucleoprotein A2/B1, cofilin 1, and peptidylprolyl isomerase A were overexpressed in tumors compared with normal tissues. In contrast, the IGHG1 protein, the complement C3 component C3c, which are two newly identified protein markers, were downregulated in IDCA tissues.

Genetically encoded proton sensors reveal activity-dependent pH changes in neurons

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Joseph Valentino Raimondo

2012-05-01

Full Text Available The regulation of hydrogen ion concentration (pH is fundamental to cell viability, metabolism and enzymatic function. Within the nervous system, the control of pH is also involved in diverse and dynamic processes including development, synaptic transmission and the control of network excitability. As pH affects neuronal activity, and can also itself be altered by neuronal activity, the existence of tools to accurately measure hydrogen ion fluctuations is important for understanding the role pH plays under physiological and pathological conditions. Outside of their use as a marker of synaptic release, genetically encoded pH sensors have not been utilised to study hydrogen ion fluxes associated with network activity. By combining whole-cell patch clamp with simultaneous two-photon or confocal imaging, we quantified the amplitude and time course of neuronal, intracellular, acidic transients evoked by epileptiform activity in two separate in vitro models of temporal lobe epilepsy. In doing so, we demonstrate the suitability of three genetically encoded pH sensors: deGFP4, E2GFP and Cl-sensor for investigating activity-dependent pH changes at the level of single neurons.

Genetically encoded proton sensors reveal activity-dependent pH changes in neurons.

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Raimondo, Joseph V; Irkle, Agnese; Wefelmeyer, Winnie; Newey, Sarah E; Akerman, Colin J

2012-01-01

The regulation of hydrogen ion concentration (pH) is fundamental to cell viability, metabolism, and enzymatic function. Within the nervous system, the control of pH is also involved in diverse and dynamic processes including development, synaptic transmission, and the control of network excitability. As pH affects neuronal activity, and can also itself be altered by neuronal activity, the existence of tools to accurately measure hydrogen ion fluctuations is important for understanding the role pH plays under physiological and pathological conditions. Outside of their use as a marker of synaptic release, genetically encoded pH sensors have not been utilized to study hydrogen ion fluxes associated with network activity. By combining whole-cell patch clamp with simultaneous two-photon or confocal imaging, we quantified the amplitude and time course of neuronal, intracellular, acidic transients evoked by epileptiform activity in two separate in vitro models of temporal lobe epilepsy. In doing so, we demonstrate the suitability of three genetically encoded pH sensors: deGFP4, E(2)GFP, and Cl-sensor for investigating activity-dependent pH changes at the level of single neurons.

Model analysis of the relationship between intracellular Po2 and energy demand in skeletal muscle

OpenAIRE

Spires, Jessica; Gladden, L. Bruce; Grassi, Bruno; Saidel, Gerald M.; Lai, Nicola

2012-01-01

On the basis of experimental studies, the intracellular O2 (iPo2)-work rate (WR) relationship in skeletal muscle is not unique. One study found that iPo2 reached a plateau at 60% of maximal WR, while another found that iPo2 decreased linearly at higher WR, inferring capillary permeability-surface area (PS) and blood-tissue O2 gradient, respectively, as alternative dominant factors for determining O2 diffusion changes during exercise. This relationship is affected by several factors, including...

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na+ and Ca2+ fluxes, Na+/K+ pump activity, and intracellular pH

International Nuclear Information System (INIS)

Mendoza, S.A.; Schneider, J.A.; Lopez-Rivas, A.; Sinnett-Smith, J.W.; Rozengurt, E.

1986-01-01

The amphibian tetradecapeptide, bombesin, and structurally related peptides caused a marked increase in ouabain-sensitive 86 Rb + uptake (a measure of Na + /K + pump activity) in quiescent Swiss 3T3 cells. This effect occurred within seconds after the addition of the peptide and appeared to be mediated by an increase in Na + entry into the cells. The effect of bombesin on Na + entry and Na + /K + pump activity was concentration dependent with half-maximal stimulation occurring at 0.3-0.4 nM. The structurally related peptides litorin, gastrin-releasing peptide, and neuromedin B also stimulated ouabain-sensitive 86 Rb + uptake; the relative potencies of these peptides in stimulating the Na + /K + pump were comparable to their potencies in increasing DNA synthesis. Bombesin increased Na + influx, at least in part, through an Na + /H + antiport. The peptide augmented intracellular pH and this effect was abolished in the absence of extracellular Na + . In addition to monovalent ion transport, bombesin and the structurally related peptides rapidly increased the efflux of 45 Ca 2+ from quiescent Swiss 3T3 cells. This Ca 2+ came from an intracellular pool and the efflux was associated with a 50% decrease in total intracellular Ca 2+ . The peptides also caused a rapid increase in cytosolic free calcium concentration. Prolonged pretreatment of Swiss 3T3 cells with phorbol dibutyrate, which causes a loss of protein kinase C activity, greatly decreased the stimulation of 86 Rb + uptake and Na + entry by bombesin implicating this phosphotransferase system in the mediation of part of these responses to bombesin. Since some activation of monovalent ion transport by bombesin was seen in phorbol dibutyrate-pretreated cells, it is likely that the peptide also stimulates monovalent ion transport by a second mechanism

Design and application of optical nanosensors for pH imaging in cell compartments

DEFF Research Database (Denmark)

Benjaminsen, Rikke Vicki; Almdal, Kristoffer

the last two decades. However, even though these sensor systems have proven themselves as superior to conventional methods, there are still questions about the use of these sensors that need to be addressed, especially regarding sensor design and calibration. We have developed a new triple-labelled p......Measurements of pH in acidic cellular compartments of mammalian cells is important for our understanding of cell metabolism, and organelle acidification is an essential event in living cells especially in the endosomal-lysosomal pathway where pH is critical for cellular sorting of internalized...... material. Intracellular pH can be measured by the use of fluorescence ratio imaging microscopy (FRIM), however, available methods for pH measurements in living cells are not optimal. Nanoparticle based optical sensor technology for quantification of metabolites in living cells has been developed over...

Effects of prolonged acclimation to cold on the extra--and intracellular acid-base status in the land snail Helix lucorum (L.).

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Staikou, A; Stiakakis, M; Michaelidis, B

2001-01-01

The aim of this study was to examine the effect of prolonged acclimation to cold on the acid-base status of extra- and intracellular fluids in the land snail Helix lucorum. For this purpose, acid-base parameters in the hemolymph and tissues were determined. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether they change in the snails during acclimation to cold. According to the results presented, there is an inverse pH-temperature relationship in the hemolymph within the first day of acclimation, which is consistent with alphastat regulation. The Pco2 decreased, and pH in the hemolymph (pH(e)) increased by 0.32 U within the first day of acclimation to cold, which corresponds to a change of 0.013 U degrees C(-1). After the first day of acclimation, Pco2 increased in the hemolymph, resulting in a significant drop in pH(e) by 90 d of acclimation to cold. Acclimation of snails to low temperatures did not change the buffer value of the hemolymph. Also, intracellular pH (pH(i)) and intracellular buffer values remained stable during acclimation to cold for prolonged periods. The latter results in conjunction with those obtained by the in vitro determination of the passive component of intracellular fluids indicate an active regulation of pH(i) in H. lucorum during acclimation to cold.

Mechanism of the negative force-frequency relationship in physiologically intact rat ventricular myocardium. Studies by intracellular Ca2+ monitor with iodo-1 and by 31P-nuclear magnetic resonance spectroscopy

International Nuclear Information System (INIS)

Morii, Isao; Kihara, Yasuki; Sasayama, Shigetake; Konishi, Takashi; Inubushi, Toshiro.

1996-01-01

We studied the subcellular mechanisms of the negative force-frequency relationship in rat myocardium by measuring intracellular Ca 2+ transients by indo-1 fluorometry and intracellular pH (pH i ) and phosphate compounds with 31 P-nuclear magnetic resonance (NMR). The data were compared with those from guinea pig hearts, which show a positive force-frequency relationship. By increasing the pacing rate from 3 Hz to 5 Hz, the peak positive first derivative of left ventricular pressure (LVdP/dt) in rat heart decreased by 10±1% (n=6). In contrast to this negative inotropic response, simultaneously measured peak Ca 2+ transients increased by 6±1%. Guinea pig heart (n=6) showed an increase in peak positive LVdP/dt (33±1%) which was associated with an increase in peak Ca 2+ transients (8±1%). Under equivalent experimental conditions in an NMR spectrometer, this increase in the pacing rate did not affect intracellular levels of phosphate compounds in either rat (n=6) or guinea pig heart (n=6). In contrast, pH i showed a decrease of 0.031±0.006 pH units in rat heart, while no changes were observed in guinea pig heart. These results suggest that in physiological rat myocardium, pH i is susceptible to changes in the stimulus frequency and may affect the Ca 2+ -responsiveness of contractile proteins, which results in the negative force-frequency relationship. (author)

Ratiometric Imaging of Extracellular pH in Dental Biofilms Using C-SNARF-4

DEFF Research Database (Denmark)

Dige, Irene

pH in dental biofilms plays a central role for the development of caries lesions. For decades, pH measurements in biofilms have been limited to recording pH with electrodes/microelectrodes that do not permit monitoring horizontal pH gradients in biofilms in real-time. Quantitative fluorescent...... microscopy can overcome these problems. Objective: The aim of this demonstration study was to monitor extracellular biofilm pH microscopically with the ratiometric pH-sensitive dye C-SNARF-4 in in-situ-grown dental biofilms. Methods: Using confocal microscopy, the dye C-SNARF-4 was employed both as p...... the microscopic images in order to exclusively determine extracellular pH. We monitored the pH drop at the biofilm-substratum interface in six microscopic fields of view per biofilm for 1h after exposure to 0.4% glucose. Results: Extracellular pH dropped rapidly in all specimens. In both individuals, analysis...

Production of citrinin-free Monascus pigments by submerged culture at low pH.

Science.gov (United States)

Kang, Biyu; Zhang, Xuehong; Wu, Zhenqiang; Wang, Zhilong; Park, Sunghoon

2014-02-05

Microbial fermentation of citrinin-free Monascus pigments is of great interest to meet the demand of food safety. In the present work, the effect of various nitrogen sources, such as monosodium glutamate (MSG), cornmeal, (NH4)₂SO₄, and NaNO₃, on Monascus fermentation was examined under different initial pH conditions. The composition of Monascus pigments and the final pH of fermentation broth after Monascus fermentation were determined. It was found that nitrogen source was directly related to the final pH and the final pH regulated the composition of Monascus pigments and the biosynthesis of citrinin. Thus, an ideal nitrogen source can be selected to control the final pH and then the citrinin biosynthesis. Citrinin-free orange pigments were produced at extremely low initial pH in the medium with (NH4)₂SO₄ or MSG as nitrogen source. No citrinin biosynthesis at extremely low pH was further confirmed by extractive fermentation of intracellular pigments in the nonionic surfactant Triton X-100 micelle aqueous solution. This is the first report about the production of citrinin-free Monascus pigments at extremely low pH. Copyright © 2013 Elsevier Inc. All rights reserved.

Integrated titanium dioxide (TiO_2) nanoparticles on interdigitated device electrodes (IDEs) for pH analysis

International Nuclear Information System (INIS)

Azizah, N.; Gopinath, Subash C. B.; Nadzirah, Sh.; Farehanim, M. A.; Fatin, M. F.; Ruslinda, A. R.; Hashim, U.; Arshad, M. K. Md.; Ayub, R. M.

2016-01-01

Titanium dioxide (TiO_2) nanoparticles based Interdigitated Device Electrodes (IDEs) Nanobiosensor device was developed for intracellular biochemical detection. Fabrication and characterization of pH sensors using IDE nanocoated with TiO_2 was studied in this paper. In this paper, a preliminary assessment of this intracellular sensor with electrical measurement under different pH levels. 3-aminopropyltriethoxysilane (APTES) was used to enhance the sensitivity of titanium dioxide layer as well as able to provide surface modification by undergoing protonation and deprotonation process. Different types of pH solution provide different resistivity and conductivity towards the surface. Base solution has the higher current compared to an acid solution. Amine and oxide functionalized TiO_2 based IDE exhibit pH-dependent could be understood in terms of the change in surface charge during protonation and deprotonation. The simple fabrication process, high sensitivity, and fast response of the TiO_2 based IDEs facilitate their applications in a wide range of areas. The small size of semiconductor TiO_2 based IDE for sensitive, label-free, real time detection of a wide range of biological species could be explored in vivo diagnostics and array-based screening.

Ph3CCOOSnPh3.Ph3PO AND Ph3CCOOSnPh3.Ph3AsO: SYNTHESIS AND INFRARED STUDY

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ABDOU MBAYE

2014-08-01

Full Text Available The mixture of ethanolic solutions of Ph3CCOOSnPh3 and Ph3PO or Ph3AsO gives Ph3CCOOSnPh3.Ph3PO and Ph3CCOOSnPh3.Ph3AsO adducts which have been characterized by infrared spectroscopy. A discrete structure is suggested for both, the environment around the tin centre being trigonal bipyramidal, the triphenylacetate anion behaving as a mondentate ligand.

Temperature dependence of intracellular Ca2+ homeostasis in rat meiotic and postmeiotic spermatogenic cells.

Science.gov (United States)

Herrera, E; Salas, K; Lagos, N; Benos, D J; Reyes, J G

2001-10-01

The hypothesis that intracellular [Ca2+] is a cell parameter responsive to extreme temperatures in rat meiotic and postmeiotic spermatogenic cells was tested using intracellular fluorescent probes for Ca2+ and pH. In agreement with this hypothesis, extreme temperatures induced a rapid increase of cytosolic [Ca2+] in rat pachytene spermatocytes and round spermatids. Oscillatory changes in temperature can induce oscillations in cytosolic [Ca2+] in these cells. Intracellular [Ca2+] homeostasis in round spermatids was more sensitive to high temperatures compared with pachytene spermatocytes. The calculated activation energies for SERCA ATPase-mediated fluxes in pachytene spermatocytes and round spermatids were 62 and 75 kJ mol(-1), respectively. The activation energies for leak fluxes from intracellular Ca2+ stores were 55 and 68 kJ mol(-1) for pachytene spermatocytes and round spermatids, respectively. Together with changes in cytosolic [Ca2+], round spermatids undergo a decrease in pH(i) at high temperatures. This temperature-induced decrease in pH(i) appears to be partially responsible for the increase in cytosolic [Ca2+] of round spermatids induced by high temperatures. This characteristic of rat meiotic and postmeiotic spermatogenic cells to undergo an increment in cytosolic Ca2+ at temperatures > 33 degrees C can be related to the induction of programmed cell death by high temperatures in these cells.

Analysis of Light-Induced Transmembrane Ion Gradients and Membrane Potential in Photosystem I Proteoliposomes

International Nuclear Information System (INIS)

Pennisi, Cristian P.; Greenbaum, Elias; Yoshida, Ken

2010-01-01

Photosystem I (PSI) complexes can support a light-driven electrochemical gradient for protons, which is the driving force for energy-conserving reactions across biological membranes. In this work, a computational model that enables a quantitative description of the light-induced proton gradients across the membrane of PSI proteoliposomes is presented. Using a set of electrodiffusion equations, a compartmental model of a vesicle suspended in aqueous medium was studied. The light-mediated proton movement was modeled as a single proton pumping step with backpressure of the electric potential. The model fits determinations of pH obtained from PSI proteoliposomes illuminated in the presence of mediators of cyclic electron transport. The model also allows analysis of the proton gradients in relation to the transmembrane ion fluxes and electric potential. Sensitivity analysis enabled a determination of the parameters that have greater influence on steady-state levels and onset/decay rates of transmembrane pH and electric potential. This model could be used as a tool for optimizing PSI proteoliposomes for photo-electrochemical applications.

The Effects of Synthetic Cannabinoids on Alveolar-Arterial Oxygen Gradient

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Egemen Kucuk

2016-09-01

Full Text Available Aim: Synthetic cannabinoids are chemicals that produce several marijuana-like effects in humans. Aim of this study is to investigate the effects of synthetic cannabinoids on to alveolar-arterial oxygen gradient. Material and Method: A total of 112 patients, who admitted directly to emergency clinic with synthetic cannabinoid usage, were determined between February 2014 and August 2014. Blood gases of 41 patients were determined as arterial blood gases on room air, and included in to study. Patients were evaluated according to age, sex, decade, partial pressure of arterial oxygen, partial pressure of arterial carbon dioxide, pH, bicarbonate, metabolic status, age consistent expected alveolar-arterial oxygen gradient and calculated alveolar-arterial oxygen gradient. Results: Synthetic cannabinoid using was higher in males, mean age of patients was 23.32±6.14 years. Number of patients in the third decade were significantly higher than the other decades. The calculated alveolar-arterial oxygen gradient value of patients was significantly higher than age consistent expected alveolar-arterial oxygen gradient value. Respiratory acidosis, was significantly higher than the other types of the metabolic disorders. The best cutoff point for calculated alveolar-arterial oxygen gradient was 12.70, with sensitivity of 90% and specifity of 85%. Area under curve was 0.70 for calculated alveolar-arterial oxygen gradient. Discussion: The value of alveolar-arterial oxygen gradient has been increased due to synthetic cannabinoid usage. This can be used as a supportive parameter in the diagnosis of synthetic cannabinoid usage.

Dependence of radioprotective effect of chemical modifying agents on their intracellular concentrations

International Nuclear Information System (INIS)

Eidus, L.K.; Korystov, Y.N.; Kublik, L.N.; Vexler, A.M.

1982-01-01

Regularities of the radioprotective effect of chemical modifying agents cysteamine, caffeine benzoate, thioglycolic acid, and caffeine, all weak electrolytes, have been studied in cultured Chinese hamster cells. Efficiency of protection is shown to be dependent on pH and concentrations of the drug inside the cells and in the medium. Based on the theory of the dissociation of weak electrolytes and their distribution between the cells and the medium a strong correlation between the efficiency of modification of the radiation response and intracellular concentration of a modifying agent is shown. (author)

Further studies on the effect of chloroquine on the uptake, metabolism and intracellular translocation of [35S]cystine in cystinotic fibroblasts.

Science.gov (United States)

Danpure, C J; Jennings, P R; Fyfe, D A

1986-03-14

The present study uses the lysosomotropic drug chloroquine to investigate the mechanisms by which exogenous [35S]cystine is able to label the intracellular (intralysosomal) cystine pool(s) in cystinotic fibroblasts. When cystinotic fibroblasts were labelled for short periods of time (8 h or less), chloroquine (20 microM) inhibited the labelling of the intracellular cystine pool(s). However, when the cells were labelled for longer periods of time (24 h or more) chloroquine stimulated the labelling of the intracellular cystine pool(s). The short-term effect was selectively abolished when the cells were washed free of chloroquine, while the long-term effect was selectively abolished when the medium was depleted of cystine. Two routes of translocation of exogenous cystine to the lysosomes could be defined. One route was fast, had a low capacity, was inhibited by chloroquine and increased with increasing medium pH, while the other route was slow, had a high capacity, was stimulated by chloroquine and was more active at low pH. The former pathway probably consisted of plasma membrane transport of cystine into the cytosol followed by direct or indirect transport into the lysosomes. The latter route possibly consisted of pinocytosis with fusion of the cystine-containing pinosomes with lysosomes.

The Final Frontier of pH and the Undiscovered Country Beyond

Science.gov (United States)

Bal, Wojciech; Kurowska, Ewa; Maret, Wolfgang

2012-01-01

The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K w = [H+]×[OH−].We used K w [in a reversed way] to calculate the number of undissociated H2O molecules required by this equilibrium constant to yield at least one of its daughter ions, H+ or OH− at a given pH. In this way we obtained a formula that relates pH to the minimal volume VpH required to provide a physical meaning to K w, (where N A is Avogadro’s number). For example, at pH 7 (neutral at 25°C) VpH = 16.6 aL. Any deviation from neutral pH results in a larger VpH value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H+ ions at equilibrium, thus the definition of pH based on K w is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H+ ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H+ ions as a general source for biochemical reactions. Consequences of this finding are discussed. PMID:23049874

How a High-Gradient Magnetic Field Could Affect Cell Life

Science.gov (United States)

Zablotskii, Vitalii; Polyakova, Tatyana; Lunov, Oleg; Dejneka, Alexandr

2016-01-01

The biological effects of high-gradient magnetic fields (HGMFs) have steadily gained the increased attention of researchers from different disciplines, such as cell biology, cell therapy, targeted stem cell delivery and nanomedicine. We present a theoretical framework towards a fundamental understanding of the effects of HGMFs on intracellular processes, highlighting new directions for the study of living cell machinery: changing the probability of ion-channel on/off switching events by membrane magneto-mechanical stress, suppression of cell growth by magnetic pressure, magnetically induced cell division and cell reprograming, and forced migration of membrane receptor proteins. By deriving a generalized form for the Nernst equation, we find that a relatively small magnetic field (approximately 1 T) with a large gradient (up to 1 GT/m) can significantly change the membrane potential of the cell and thus have a significant impact on not only the properties and biological functionality of cells but also cell fate. PMID:27857227

How a High-Gradient Magnetic Field Could Affect Cell Life

Science.gov (United States)

Zablotskii, Vitalii; Polyakova, Tatyana; Lunov, Oleg; Dejneka, Alexandr

2016-11-01

The biological effects of high-gradient magnetic fields (HGMFs) have steadily gained the increased attention of researchers from different disciplines, such as cell biology, cell therapy, targeted stem cell delivery and nanomedicine. We present a theoretical framework towards a fundamental understanding of the effects of HGMFs on intracellular processes, highlighting new directions for the study of living cell machinery: changing the probability of ion-channel on/off switching events by membrane magneto-mechanical stress, suppression of cell growth by magnetic pressure, magnetically induced cell division and cell reprograming, and forced migration of membrane receptor proteins. By deriving a generalized form for the Nernst equation, we find that a relatively small magnetic field (approximately 1 T) with a large gradient (up to 1 GT/m) can significantly change the membrane potential of the cell and thus have a significant impact on not only the properties and biological functionality of cells but also cell fate.

Integrating chemical imaging of cationic trace metal solutes and pH into a single hydrogel layer

Energy Technology Data Exchange (ETDEWEB)

Hoefer, Christoph [Department of Forest and Soil Sciences, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Konrad-Lorenz-Strasse 24, A-3430 Tulln (Austria); Santner, Jakob, E-mail: jakob.santner@boku.ac.at [Department of Forest and Soil Sciences, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Konrad-Lorenz-Strasse 24, A-3430 Tulln (Austria); Department of Crop Sciences, Division of Agronomy, University of Natural Resources and Life Sciences, Vienna, Konrad-Lorenz-Strasse 24, 3430 Tulln (Austria); Borisov, Sergey M. [Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010, Graz (Austria); Wenzel, Walter W.; Puschenreiter, Markus [Department of Forest and Soil Sciences, Institute of Soil Research, University of Natural Resources and Life Sciences, Vienna, Konrad-Lorenz-Strasse 24, A-3430 Tulln (Austria)

2017-01-15

Gel-based, two-dimensional (2D) chemical imaging techniques are versatile methods for investigating biogeochemically active environments at high spatial resolution (sub-mm). State-of-the-art solute imaging techniques, such as diffusive gradients in thin films (DGT) and planar optodes (PO), employ passive solute sampling or sensing. Combining these methods will provide powerful tools for studying the biogeochemistry of biological niches in soils and sediments. In this study we aimed at developing a combined single-layer gel for direct pH imaging using PO and sampling of anionic and cationic solutes by DGT, with subsequent analysis of the bound solutes by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). We tested three ultra-thin (<100 μm) polyurethane-based gels, incorporating anion and cation binding materials and the fluorescent pH indicator DCIFODA (2′,7′-dichloro-5(6)-N-octadecyl-carboxamidofluorescein). Results showed that PO-based pH sensing using DCIFODA was impossible in the presence of the anion binding materials due to interferences with DCIFODA protonation. One gel, containing only a cation binding material and DCIFODA, was fully characterized and showed similar performance characteristics as comparable DGT-only gels (applicable pH range: pH 5–8, applicable ionic strength range: 1–20 mmol L{sup -1}, cation binding capacity ∼24 μg cm{sup −2}). The dynamic range for PO-based pH mapping was between pH 5.5 and 7.5 with t{sub 90} response time of ∼60 min. In a case study we demonstrated the gel's suitability for multi-analyte solute imaging and mapped pH gradients and concurrent metal solubility patterns in the rhizosphere of Salix smithiana. pH decreases in the rooted soil were co-localized with elevated solute fluxes of Al{sup 3+}, Co{sup 2+}, Cu{sup 2+}, Fe, Mn{sup 2+}, Ni{sup 2+} and Pb{sup 2+}, indicating pH-induced metal solubilisation. - Highlights: • Diffusive gradients in thin films (DGT) and planar

Integrating chemical imaging of cationic trace metal solutes and pH into a single hydrogel layer

International Nuclear Information System (INIS)

Hoefer, Christoph; Santner, Jakob; Borisov, Sergey M.; Wenzel, Walter W.; Puschenreiter, Markus

2017-01-01

Gel-based, two-dimensional (2D) chemical imaging techniques are versatile methods for investigating biogeochemically active environments at high spatial resolution (sub-mm). State-of-the-art solute imaging techniques, such as diffusive gradients in thin films (DGT) and planar optodes (PO), employ passive solute sampling or sensing. Combining these methods will provide powerful tools for studying the biogeochemistry of biological niches in soils and sediments. In this study we aimed at developing a combined single-layer gel for direct pH imaging using PO and sampling of anionic and cationic solutes by DGT, with subsequent analysis of the bound solutes by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). We tested three ultra-thin (<100 μm) polyurethane-based gels, incorporating anion and cation binding materials and the fluorescent pH indicator DCIFODA (2′,7′-dichloro-5(6)-N-octadecyl-carboxamidofluorescein). Results showed that PO-based pH sensing using DCIFODA was impossible in the presence of the anion binding materials due to interferences with DCIFODA protonation. One gel, containing only a cation binding material and DCIFODA, was fully characterized and showed similar performance characteristics as comparable DGT-only gels (applicable pH range: pH 5–8, applicable ionic strength range: 1–20 mmol L"-"1, cation binding capacity ∼24 μg cm"−"2). The dynamic range for PO-based pH mapping was between pH 5.5 and 7.5 with t_9_0 response time of ∼60 min. In a case study we demonstrated the gel's suitability for multi-analyte solute imaging and mapped pH gradients and concurrent metal solubility patterns in the rhizosphere of Salix smithiana. pH decreases in the rooted soil were co-localized with elevated solute fluxes of Al"3"+, Co"2"+, Cu"2"+, Fe, Mn"2"+, Ni"2"+ and Pb"2"+, indicating pH-induced metal solubilisation. - Highlights: • Diffusive gradients in thin films (DGT) and planar optode (PO) imaging is combined. • A

Fluorescent nanosensors for intracellular measurements: synthesis, characterisation, calibration and measurement

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Arpan Shailesh Desai

2014-01-01

Full Text Available Measurement of intracellular acidification is important for understanding fundamental biological pathways as well as developing effective therapeutic strategies. Fluorescent pH nanosensors are an enabling technology for real-time monitoring of intracellular acidification. The physicochemical characteristics of nanosensors can be engineered to target specific cellular compartments and respond to external stimuli. Therefore nanosensors represent a versatile approach for probing biological pathways inside cells. The fundamental components of nanosensors comprise a pH-sensitive fluorophore (signal transducer and a pH-insensitive reference fluorophore (internal standard immobilised in an inert non-toxic matrix. The inert matrix prevents interference of cellular components with the sensing elements as well as minimizing potentially harmful effects of some fluorophores on cell function. Fluorescent nanosensors are synthesised using standard laboratory equipment and are detectable by non-invasive widely accessibly imaging techniques. The outcomes of studies employing this technology are dependent on reliable methodology for performing measurements. In particular special consideration must be given to conditions for sensor calibration, uptake conditions and parameters for image analysis. We describe procedures for: 1 synthesis and characterisation of polyacrylamide and silica based nanosensors 2 nanosensor calibration and 3 performing measurements using fluorescence microscopy.

Quantification of Radiation-induced DNA Damage following intracellular Auger-Cascades

DEFF Research Database (Denmark)

Fredericia, Nina Pil Møntegaard

2017-01-01

Purpose: The aim my PhD study and the topic of this thesis is to investigate the radiotoxicity and the Relative Biological effectiveness (RBE) of intracellular Auger cascades. A special focus is kept on obtaining reliable absorbed dose calculations and using matched dose rate profiles for the Auger......-values (SC-values). The work can be divided into three steps; Examination of the bio-kinetics of the Auger emitter 131Cs used in the study, calculations of the SC-values and finally the measurement of the RBE of intracellular 131Cs decays, through ƴH2AX and clonogenic cell survival assay. Methods: A series....../(Bq*Sec)/pL for HeLa nuclei and from 7.45*10-4 to 7.63 *10-4 Gy/(Bq*Sec)/pL for V79 nuclei. The SC-values were shown to be were very robust and almost independent of cellular and nuclear size. A RBE value of 1 was obtained for HeLa cells using ƴH2AX assays. RBE values of 4.5 ± 0.5 and 3.8 ± 0.8 were obtained for He...

Subtype-specific, bi-component inhibition of SK channels by low internal pH

DEFF Research Database (Denmark)

Peitersen, Torben; Jespersen, Thomas; Jorgensen, Nanna K

2006-01-01

The effects of low intracellular pH (pH(i) 6.4) on cloned small-conductance Ca2+-activated K+ channel currents of all three subtypes (SK1, SK2, and SK3) were investigated in HEK293 cells using the patch-clamp technique. In 400 nM internal Ca2+ [Ca2+]i, all subtypes were inhibited by pH(i) 6...

SITS-sensitive Cl- conductance pathway in chick intestinal cells

International Nuclear Information System (INIS)

Montrose, M.; Randles, J.; Kimmich, G.A.

1987-01-01

The unidirectional influx of 36 Cl - into isolated chick epithelial cells is 30% inhibited by 300 μM SITS. Characteristics of the SITS-sensitive flux pathway were examined in terms of sensitivity to changes in membrane potential and intracellular pH. Potential dependence was evaluated using unidirectional influx of [ 14 C]tetraphenylphosphonium ([ 14 C]-TPP + ) as a qualitative sensor of diffusion potentials created by experimentally imposed gradients of CL - . Steady-state distribution of [ 14 C]methylamine ([ 14 C]MA) was used to examine for Cl - -dependent changes in intracellular pH. Imposed Na + gradients, but not Cl - gradients, induce changes in [ 14 C]MA distribution. SITS does not alter the [ 14 C]MA distribution observed in cells with imposed gradients of Na + and Cl - . Both results suggest that inhibition of Cl - influx. However, if relative permeabilities for ion pairs via conductance pathways are compared, it can be shown that SITS causes a marked reduction of P Cl relative to either P Na or P K . SITS also inhibits electrically induced influx of [ 14 C]TPP + or [ 14 C]α-methylglucoside driven by imposed Cl - influx can be blocked by SITS. These observations are all consistent with a SITS-sensitive Cl - conductance pathway associated with the plasma membrane of chick intestinal cells. No Cl - -OH - exchange capability can be detected for chick intestinal cells

Phytosynthesis of intracellular and extracellular gold nanoparticles by living peanut plant (Arachis hypogaea L.).

Science.gov (United States)

Raju, Dugyala; Mehta, Urmil J; Ahmad, Absar

2012-01-01

Inorganic nanomaterials of different chemical compositions are conventionally synthesized under harsh environments such as extremes of temperature, pressure, and pH. Moreover, these methods are eco-unfriendly and cumbersome, yield bigger particles, and agglomerate because of not being capped by capping agents. In contrast, biological synthesis of inorganic nanomaterials occurs under ambient conditions, namely room temperature, atmospheric pressure, and physiological pH. These methods are reliable, eco-friendly, and cheap. In this paper, we report for the first time the extracellular and intracellular synthesis of gold nanoparticles (GNPs) using living peanut seedlings. The formed GNPs were highly stable in solution and inside the plant tissue. Transmission electron microscopy revealed that extracellular GNPs distributions were in the form of monodispersed nanoparticles. The nanoparticles ranged from 4 to 6 nm in size. The intercellular nanoparticles were of oval shape and size ranged from 5 to 50 nm. Both extracellular and intracellular nanoparticles were further characterized by standard techniques. The formed GNPs inside the plant tissue were estimated by inductively coupled plasma spectrometry. This opens up an exciting possibility of a plant-based nanoparticle synthesis strategy, wherein the nanoparticles may be entrapped in the biomass in the form of a film or produced in the solution, both of which have interesting applications. © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Detection of Metabolic Fluxes of O and H Atoms into Intracellular Water in Mammalian Cells

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Kreuzer, Helen W.; Quaroni, Luca; Podlesak, David W.; Zlateva, Theodora; Bollinger, Nikki; McAllister, Aaron; Lott, Michael J.; Hegg, Eric L.

2012-01-01

Metabolic processes result in the release and exchange of H and O atoms from organic material as well as some inorganic salts and gases. These fluxes of H and O atoms into intracellular water result in an isotopic gradient that can be measured experimentally. Using isotope ratio mass spectroscopy, we revealed that slightly over 50% of the H and O atoms in the intracellular water of exponentially-growing cultured Rat-1 fibroblasts were isotopically distinct from growth medium water. We then employed infrared spectromicroscopy to detect in real time the flux of H atoms in these same cells. Importantly, both of these techniques indicate that the H and O fluxes are dependent on metabolic processes; cells that are in lag phase or are quiescent exhibit a much smaller flux. In addition, water extracted from the muscle tissue of rats contained a population of H and O atoms that were isotopically distinct from body water, consistent with the results obtained using the cultured Rat-1 fibroblasts. Together these data demonstrate that metabolic processes produce fluxes of H and O atoms into intracellular water, and that these fluxes can be detected and measured in both cultured mammalian cells and in mammalian tissue. PMID:22848359

Acidic pH sensing in the bacterial cytoplasm is required for Salmonella virulence.

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Choi, Jeongjoon; Groisman, Eduardo A

2016-09-01

pH regulates gene expression, biochemical activities and cellular behaviors. A mildly acidic pH activates the master virulence regulatory system PhoP/PhoQ in the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The sensor PhoQ harbors an extracytoplasmic domain implicated in signal sensing, and a cytoplasmic domain controlling activation of the regulator PhoP. We now report that, surprisingly, a decrease in Salmonella's own cytoplasmic pH induces transcription of PhoP-activated genes even when the extracytoplasmic pH remains neutral. Amino acid substitutions in PhoQ's cytoplasmic domain hindered activation by acidic pH and attenuated virulence in mice, but did not abolish activation by low Mg(2+) or the antimicrobial peptide C18G. Conversely, removal of PhoQ's extracytoplasmic domains prevented the response to the latter PhoQ-activating signals but not to acidic pH. PhoP-dependent genes were minimally induced by acidic pH in the non-pathogenic species Salmonella bongori but were activated by low Mg(2+) and C18G as in pathogenic S. enterica. Our findings indicate that the sensor PhoQ enables S. enterica to respond to both host- and bacterial-derived signals that alter its cytoplasmic pH. © 2016 John Wiley & Sons Ltd.

Effect of pH on molecular constitution and distribution of hemoglobin in living erythrocyte.

Science.gov (United States)

Wu, Yue; Huang, Yao-Xiong; Kang, Li-Li; Wu, Zheng-Jie; Luo, Man

2010-04-01

The molecular constitution of in situ hemoglobin (Hb) and their distribution in living erythrocyte were investigated versus pH using the technique of confocal Raman microscopy. Both Raman point spectra and line mapping measurements were performed on living erythrocytes in suspensions with pH values from 4.82 to 9.70. It was found that the Hb inside a living erythrocyte would dissociate into monomer/dimer when the cells are in low and high pH environments. In contrast to the homogeneous distribution of the Hbs in the cells in neutral suspension, there are more Hbs distributing around the cell membrane or binding to the membrane as pH increases. While in low pH, as the cell become spherical, most of the Hbs distribute to the central part of the cell. In summary, our investigation suggests that the variation of the external pH not only brings changes in the morphology and membrane structure of an erythrocyte, but also affects the constitution and distribution of its intracellular Hbs, thereby the flexibility of the cell membrane and the oxygenation ability of the Hb.

Self-Assembled Fluorescent Bovine Serum Albumin Nanoprobes for Ratiometric pH Measurement inside Living Cells.

Science.gov (United States)

Yang, Qiaoyu; Ye, Zhongju; Zhong, Meile; Chen, Bo; Chen, Jian; Zeng, Rongjin; Wei, Lin; Li, Hung-wing; Xiao, Lehui

2016-04-20

In this work, we demonstrated a new ratiometric method for the quantitative analysis of pH inside living cells. The structure of the nanosensor comprises a biofriendly fluorescent bovine serum albumin (BSA) matrix, acting as a pH probe, and pH-insensitive reference dye Alexa 594 enabling ratiometric quantitative pH measurement. The fluorescent BSA matrix was synthesized by cross-linking of the denatured BSA proteins in ethanol with glutaraldehyde. The size of the as-synthesized BSA nanoparticles can be readily manipulated from 30 to 90 nm, which exhibit decent fluorescence at the peak wavelength of 535 nm with a pH response range of 6-8. The potential of this pH sensor for intracellular pH monitoring was demonstrated inside living HeLa cells, whereby a significant change in fluorescence ratio was observed when the pH of the cell was switched from normal to acidic with anticancer drug treatment. The fast response of the nanosensor makes it a very powerful tool in monitoring the processes occurring within the cytosol.

Reversibly extracellular pH controlled cellular uptake and photothermal therapy by PEGylated mixed-charge gold nanostars.

Science.gov (United States)

Wang, Shouju; Teng, Zhaogang; Huang, Peng; Liu, Dingbin; Liu, Ying; Tian, Ying; Sun, Jing; Li, Yanjun; Ju, Huangxian; Chen, Xiaoyuan; Lu, Guangming

2015-04-17

Shielding nanoparticles from nonspecific interactions with normal cells/tissues before they reach and after they leave tumors is crucial for the selective delivery of NPs into tumor cells. By utilizing the reversible protonation of weak electrolytic groups to pH changes, long-chain amine/carboxyl-terminated polyethylene glycol (PEG) decorated gold nanostars (GNSs) are designed, exhibiting reversible, significant, and sensitive response in cell affinity and therapeutic efficacy to the extracellular pH (pHe) gradient between normal tissues and tumors. This smart nanosystem shows good dispersity and unimpaired photothermal efficacy in complex bioenvironment at pH 6.4 and 7.4 even when their surface charge is neutral. One PEGylated mixed-charge GNSs with certain surface composition, GNS-N/C 4, exhibits high cell affinity and therapeutic efficacy at pH 6.4, and low affinity and almost "zero" damage to cells at pH 7.4. Remarkably, this significant and sensitive response in cell affinity and therapeutic efficacy is reversible as local pH alternated. In vivo, GNS-N/C 4 shows higher accumulation in tumors and improved photothermal therapeutic efficacy than pH-insensitive GNSs. This newly developed smart nanosystem, whose cell affinity reversibly transforms in response to pHe gradient with unimpaired biostability, provides a novel effective means of tumor-selective therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Contribution of pH, diprotonated phosphate and potassium for the reflex increase in blood pressure during handgrip

DEFF Research Database (Denmark)

Boushel, Robert Christopher; Madsen, P; Nielsen, H B

1998-01-01

The relative importance of pH, diprotonated phosphate (H2PO4-) and potassium (K+) for the reflex increase in mean arterial pressure (MAP) during exercise was evaluated in seven subjects during rhythmic handgrip at 15 and 30% maximal voluntary contraction (MVC), followed by post-exercise muscle...... to the exercise levels. Analysis of each variable as a predictor of blood pressure indicated that only the intracellular pH and diprotonated phosphate were linked to the reflex elevation of blood pressure during handgrip....

TRPM5 mediates acidic extracellular pH signaling and TRPM5 inhibition reduces spontaneous metastasis in mouse B16-BL6 melanoma cells.

Science.gov (United States)

Maeda, Toyonobu; Suzuki, Atsuko; Koga, Kaori; Miyamoto, Chihiro; Maehata, Yojiro; Ozawa, Shigeyuki; Hata, Ryu-Ichiro; Nagashima, Yoji; Nabeshima, Kazuki; Miyazaki, Kaoru; Kato, Yasumasa

2017-10-03

Extracellular acidity is a hallmark of solid tumors and is associated with metastasis in the tumor microenvironment. Acidic extracellular pH (pH e ) has been found to increase intracellular Ca 2+ and matrix metalloproteinase-9 (MMP-9) expression by activating NF-κB in the mouse B16 melanoma model. The present study assessed whether TRPM5, an intracellular Ca 2+ -dependent monovalent cation channel, is associated with acidic pH e signaling and induction of MMP-9 expression in this mouse melanoma model. Treatment of B16 cells with Trpm5 siRNA reduced acidic pH e -induced MMP-9 expression. Enforced expression of Trpm5 increased the rate of acidic pH e -induced MMP-9 expression, as well as increasing experimental lung metastasis. This genetic manipulation did not alter the pH e critical for MMP-9 induction but simply amplified the percentage of inducible MMP-9 at each pH e . Treatment of tumor bearing mice with triphenylphosphine oxide (TPPO), an inhibitor of TRPM5, significantly reduced spontaneous lung metastasis. In silico analysis of clinical samples showed that high TRPM5 mRNA expression correlated with poor overall survival rate in patients with melanoma and gastric cancer but not in patients with cancers of the ovary, lung, breast, and rectum. These results showed that TRPM5 amplifies acidic pH e signaling and may be a promising target for preventing metastasis of some types of tumor.

Relations between pH, oxygen partial pressure and growth in cultured cell spheroids.

Science.gov (United States)

Carlsson, J; Acker, H

1988-11-15

The pH gradients, oxygen partial-pressure gradients and growth curves were measured for 7 different types of spheroids. Growth curves were measured in liquid overlay culture and thereafter the spheroids were attached to cover glasses and transferred to a chamber for micro-electrode measurements. The spheroids were randomly divided for pH or pO2 measurements which then were made under conditions as identical as possible. The decreases in pO2 and pH, delta pO2 and delta pH were calculated as the difference between the values in the culture medium and the values 200 micron inside the spheroids. Each type of spheroid had a certain relation between delta pO2 and delta pH. The human colon carcinoma HT29, the mouse mammary carcinoma EMT6 and the hamster lung V79-379A spheroids had high values of the quotient delta pO2/delta pH. The human thyroid carcinoma HTh7 spheroids and the 3 types of human glioma spheroids had lower quotients. There was a tendency for fast-growing spheroids to have high quotients. Two extreme types of spheroids, HT29 (high quotient) and U-118 MG (low quotient) were analyzed for lactate production and oxygen consumption. The U-118 MG spheroids produced about 3 times more lactate and consumed about 3 times less oxygen than the HT29 spheroids. The differences in lactate production could not be explained by differences in the pyruvate Km values of lactate dehydrogenase. The results indicate that there are significant metabolic differences between the spheroid systems studied.

Inhibition of Candida albicans by Fluvastatin Is Dependent on pH

Directory of Open Access Journals (Sweden)

Martin Schmidt

2009-01-01

Full Text Available The cholesterol-lowering drug fluvastatin (FS has an inhibitory effect on the growth of the pathogenic yeast Candida albicans that is dependent on the pH of the medium. At the low pH value of the vagina, FS is growth inhibitory at low and at high concentrations, while at intermediate concentrations (1–10 mM, it has no inhibitory effect. Examination of the effect of the common antifungal drug fluconazole in combination with FS demonstrates drug interactions in the low concentration range. Determination of intracellular stress and the activity of the FS target enzyme HMG-CoA reductase confirm our hypothesis that in the intermediate dose range adjustments to the sterol biosynthesis pathway can compensate for the action of FS. We conclude that the pH dependent uptake of FS across yeast membranes might make FS combination therapy an attractive possibility for treatment of vaginal C. albicans infections.

The final frontier of pH and the undiscovered country beyond.

Directory of Open Access Journals (Sweden)

Wojciech Bal

Full Text Available The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K(w = [H(+]×[OH(-].We used K(w [in a reversed way] to calculate the number of undissociated H(2O molecules required by this equilibrium constant to yield at least one of its daughter ions, H(+ or OH(- at a given pH. In this way we obtained a formula that relates pH to the minimal volume V(pH required to provide a physical meaning to K(w, V(pH=10(pH-pK(w/2 x 10(pK(w/2/N(A (where N(A is Avogadro's number. For example, at pH 7 (neutral at 25°C V(pH =16.6 aL. Any deviation from neutral pH results in a larger V(pH value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H(+ ions at equilibrium, thus the definition of pH based on K(w is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H(+ ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H(+ ions as a general source for biochemical reactions. Consequences of this finding are discussed.

Smart Nanoparticles Undergo Phase Transition for Enhanced Cellular Uptake and Subsequent Intracellular Drug Release in a Tumor Microenvironment.

Science.gov (United States)

Ye, Guihua; Jiang, Yajun; Yang, Xiaoying; Hu, Hongxiang; Wang, Beibei; Sun, Lu; Yang, Victor C; Sun, Duxin; Gao, Wei

2018-01-10

Inefficient cellular uptake and intracellular drug release at the tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle that undergoes phase transition in a tumor microenvironment (TME). The smart nanoparticle is generated using a lipid-polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH-sensitive hydrophobic poly-l-histidine core and is loaded with the antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase transition at two different pH values in the TME: (i) At the TME (pH e : 7.0-6.5), the smart nanoparticle swells, and its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at the acid endolysosome (pH endo : 6.5-4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into the cytoplasm. In addition, a tumor-penetrating peptide iNRG was modified on the surface of the smart nanoparticle as a tumor target moiety. The in vitro studies demonstrated that the iNGR-modified smart nanoparticles promoted cellular uptake in the acidic environment (pH 6.8). The in vivo studies showed that the iNGR-modified smart nanoparticles exerted more potent antitumor efficacy against late-stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also have advantages in the future manufacture for clinical trials and clinical use.

Multi-color incomplete Cholesky conjugate gradient methods for vector computers. Ph.D. Thesis

Science.gov (United States)

Poole, E. L.

1986-01-01

In this research, we are concerned with the solution on vector computers of linear systems of equations, Ax = b, where A is a larger, sparse symmetric positive definite matrix. We solve the system using an iterative method, the incomplete Cholesky conjugate gradient method (ICCG). We apply a multi-color strategy to obtain p-color matrices for which a block-oriented ICCG method is implemented on the CYBER 205. (A p-colored matrix is a matrix which can be partitioned into a pXp block matrix where the diagonal blocks are diagonal matrices). This algorithm, which is based on a no-fill strategy, achieves O(N/p) length vector operations in both the decomposition of A and in the forward and back solves necessary at each iteration of the method. We discuss the natural ordering of the unknowns as an ordering that minimizes the number of diagonals in the matrix and define multi-color orderings in terms of disjoint sets of the unknowns. We give necessary and sufficient conditions to determine which multi-color orderings of the unknowns correpond to p-color matrices. A performance model is given which is used both to predict execution time for ICCG methods and also to compare an ICCG method to conjugate gradient without preconditioning or another ICCG method. Results are given from runs on the CYBER 205 at NASA's Langley Research Center for four model problems.

Nitrogen dynamics in oak forest soils along a historical deposition gradient

Science.gov (United States)

Ralph E. J. Boerner; Elaine Kennedy Sutherland

1995-01-01

This study quantified soil nutrient status and N mineralization/nitrification potentials in soils of oakdominated, unmanaged forest stands in seven experimental forests ranging along a historical and current acidic deposition gradient from southern Illinois to central West Virginia, U.S.A. Among these seven sites (that spanned 8.5º of longitude) soil pH and Ca...

A pH-Sensing Optode for Mapping Spatiotemporal Gradients in 3D Paper-Based Cell Cultures.

Science.gov (United States)

Kenney, Rachael M; Boyce, Matthew W; Whitman, Nathan A; Kromhout, Brenden P; Lockett, Matthew R

2018-02-06

Paper-based cultures are an emerging platform for preparing 3D tissue-like structures. Chemical gradients can be imposed upon these cultures, generating microenvironments similar to those found in poorly vascularized tumors. There is increasing evidence that the tumor microenvironment is responsible for promoting drug resistance and increased invasiveness. Acidosis, or the acidification of the extracellular space, is particularly important in promoting these aggressive cancer phenotypes. To better understand how cells respond to acidosis there is a need for 3D culture platforms that not only model relevant disease states but also contain sensors capable of quantifying small molecules in the extracellular environment. In this work, we describe pH-sensing optodes that are capable of generating high spatial and temporal resolution maps of pH gradients in paper-based cultures. This sensor was fabricated by suspending microparticles containing pH-sensitive (fluorescein) and pH-insensitive (diphenylanthracene) dyes in a polyurethane hydrogel, which was then coated onto a transparent film. The pH-sensing films have a fast response time, are reversible, stable in long-term culture environments, have minimal photobleaching, and are not cytotoxic. These films have a pK a of 7.61 ± 0.04 and are sensitive in the pH range corresponding to normal and tumorigenic tissues. With these optodes, we measured the spatiotemporal evolution of pH gradients in paper-based tumor models.

Effect of systemic pH on pHi and lactic acid generation in exhaustive forearm exercise

International Nuclear Information System (INIS)

Hood, V.L.; Schubert, C.; Keller, U.; Mueller, S.

1988-01-01

To investigate whether changes in systemic pH affect intracellular pH (pH i ), energy-rich phosphates, and lactic acid generation in muscle, eight normal volunteers performed exhaustive forearm exercise with arterial blood flow occluded for 2 min on three occasions. Subjects ingested 4 mmol/kg NH 4 Cl (acidosis; A) or NaHCO 3 (alkalosis; B) or nothing (control; C) 3 h before the exercise. Muscle pH i and phosphocreatine (PCr) content were measured with 31 P-nuclear magnetic resonance ( 31 P-NMR) spectroscopy during exercise and recovery. Lactate output during 0.5-7 min of recovery was calculated as deep venous-arterial concentration differences times forearm blood flow. Before exercise, blood pH and bicarbonate were lower in acidosis than alkalosis and intermediate in control. Lactic acid output during recovery was less with A than B and intermediate in C. PCr utilization and resynthesis were not affected by extracellular pH changes. pH i did not differ before exercise or at its end. Hence systemic acidosis inhibited and alkalosis stimulated lactic acid output. These findings suggest that systemic pH regulates cellular acid production, protecting muscle pH, at the expense of energy availability

PH Measurements of the Brain Using Phosphorus Magnetic Resonance Spectroscopy (31PMRS) in Healthy Men – Comparison of Two Analysis Methods

International Nuclear Information System (INIS)

Cichocka, Monika; Kozub, Justyna; Urbanik, Andrzej

2015-01-01

Intracellular pH provides information on homeostatic mechanisms in neurons and glial cells. The aim of this study was to define pH of the brain of male volunteers using phosphorus magnetic resonance spectroscopy ( 31 PMRS) and to compare two methods of calculating this value. In this study, 35 healthy, young, male volunteers (mean age: 25 years) were examined by 31 PMRS in 1.5 T MR system (Signa Excite, GE). The FID CSI (Free Induction Decay Chemical Shift Imaging) sequence was used with the following parameters: TR=4000 ms, FA=90°, NEX=2. Volume of interest (VOI) was selected depending on the size of the volunteers’ brain (11–14 cm 3 , mean 11.53 cm 3 ). Raw data were analyzed using SAGE (GE) software. Based on the chemical shift of peaks in the 31 PMRS spectrum, intracellular pH was calculated using two equations. In both methods the mean pH was slightly alkaline (7.07 and 7.08). Results were compared with a t-test. Significant difference (p<0.05) was found between these two methods. The 31 PMRS method enables non-invasive in vivo measurements of pH. The choice of the calculation method is crucial for computing this value. Comparing the results obtained by different teams can be done in a fully credible way only if the calculations were performed using the same formula

Comparison of Salivary pH, Buffering Capacity and Alkaline Phosphatase in Smokers and Healthy Non-Smokers: Retrospective cohort study

OpenAIRE

Ahmadi-Motamayel, Fatemeh; Falsafi, Parisa; Goodarzi, Mohammad T.; Poorolajal, Jalal

2016-01-01

Objectives: Saliva contains alkaline phosphatase (ALP)—a key intracellular enzyme related to destructive processes and cellular damage—and has buffering capacity (BC) against acids due to the presence of bicarbonate and phosphate ions. Smoking may have deleterious effects on the oral environment due to pH changes which can affect ALP activity. This study aimed to evaluate the salivary pH, BC and ALP activity of male smokers and healthy non-smokers. Methods: This retrospective cohort study ...

Fungal Communities and Functional Guilds Shift Along an Elevational Gradient in the Southern Appalachian Mountains.

Science.gov (United States)

Veach, Allison M; Stokes, C Elizabeth; Knoepp, Jennifer; Jumpponen, Ari; Baird, Richard

2017-12-04

Nitrogen deposition alters forest ecosystems particularly in high elevation, montane habitats where nitrogen deposition is greatest and continues to increase. We collected soils across an elevational (788-1940 m) gradient, encompassing both abiotic (soil chemistry) and biotic (vegetation community) gradients, at eight locations in the southern Appalachian Mountains of southwestern North Carolina and eastern Tennessee. We measured soil chemistry (total N, C, extractable PO 4 , soil pH, cation exchange capacity [ECEC], percent base saturation [% BS]) and dissected soil fungal communities using ITS2 metabarcode Illumina MiSeq sequencing. Total soil N, C, PO 4 , % BS, and pH increased with elevation and plateaued at approximately 1400 m, whereas ECEC linearly increased and C/N decreased with elevation. Fungal communities differed among locations and were correlated with all chemical variables, except PO 4 , whereas OTU richness increased with total N. Several ecological guilds (i.e., ectomycorrhizae, saprotrophs, plant pathogens) differed in abundance among locations; specifically, saprotroph abundance, primarily attributable to genus Mortierella, was positively correlated with elevation. Ectomycorrhizae declined with total N and soil pH and increased with total C and PO 4 where plant pathogens increased with total N and decreased with total C. Our results demonstrate significant turnover in taxonomic and functional fungal groups across elevational gradients which facilitate future predictions on forest ecosystem change in the southern Appalachians as nitrogen deposition rates increase and regional temperature and precipitation regimes shift.

Bacterial profile of dentine caries and the impact of pH on bacterial population diversity.

Directory of Open Access Journals (Sweden)

Nima Kianoush

Full Text Available Dental caries is caused by the release of organic acids from fermentative bacteria, which results in the dissolution of hydroxyapatite matrices of enamel and dentine. While low environmental pH is proposed to cause a shift in the consortium of oral bacteria, favouring the development of caries, the impact of this variable has been overlooked in microbial population studies. This study aimed to detail the zonal composition of the microbiota associated with carious dentine lesions with reference to pH. We used 454 sequencing of the 16S rRNA gene (V3-V4 region to compare microbial communities in layers ranging in pH from 4.5-7.8 from 25 teeth with advanced dentine caries. Pyrosequencing of the amplicons yielded 449,762 sequences. Nine phyla, 97 genera and 409 species were identified from the quality-filtered, de-noised and chimera-free sequences. Among the microbiota associated with dentinal caries, the most abundant taxa included Lactobacillus sp., Prevotella sp., Atopobium sp., Olsenella sp. and Actinomyces sp. We found a disparity between microbial communities localised at acidic versus neutral pH strata. Acidic conditions were associated with low diversity microbial populations, with Lactobacillus species including L. fermentum, L. rhamnosus and L. crispatus, being prominent. In comparison, the distinctive species of a more diverse flora associated with neutral pH regions of carious lesions included Alloprevotella tanerrae, Leptothrix sp., Sphingomonas sp. and Streptococcus anginosus. While certain bacteria were affected by the pH gradient, we also found that ∼ 60% of the taxa associated with caries were present across the investigated pH range, representing a substantial core. We demonstrated that some bacterial species implicated in caries progression show selective clustering with respect to pH gradient, providing a basis for specific therapeutic strategies.

Intracellular fate of recombinant human interferon-gamma (rIFN) in U937 cells

International Nuclear Information System (INIS)

Finbloom, D.S.

1986-01-01

After IFN binds to specific receptors on macrophages, both modulation of surface molecules and induction of microbicidal and tumoricidal activity occurs 24-48 hr later. Since the intracellular events required to insure these responses are poorly defined, the fate of radiolabeled rIFN in U937 cells was examined. Endocytosis was determined by exposing cells to pH 2.5 to allow rIFN to dissociate leaving only intracellular ligand. Degradation was measured as trichloroacetic acid soluble radioactivity in the media. Of the 4-5000 molecules of rIFN that specifically and saturably (at 300 U/ml) bound at 4 0 C, 40% dissociated during 15-30 min after cells were warmed to 37 0 C. However, if cells were continuously exposed at 37 0 C to lower levels of rIFN (60-100 U/ml), 30-40% of those molecules capable of binding to the cell at that concentration were internalized. Furthermore, 60% of the molecules were degraded during 3-4 hr of additional culture. Since exposure of cells to chloroquine and monensin resulted in only partial inhibition of degradation (75% and 43%, respectively), there may also be degradation within endosomes or on the cell following binding to its receptor. Soon thereafter, degradation products are measurable. Since many biological responses require prolonged incubation with the molecule, intracellular processing of IFN may be important for expression of these effects

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

Science.gov (United States)

Khan, Meraj A.; Philip, Lijy M.; Cheung, Guillaume; Vadakepeedika, Shawn; Grasemann, Hartmut; Sweezey, Neil; Palaniyar, Nades

2018-01-01

Neutrophils migrating from the blood (pH 7.35–7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

Directory of Open Access Journals (Sweden)

Meraj A. Khan

2018-02-01

Full Text Available Neutrophils migrating from the blood (pH 7.35–7.45 into the surrounding tissues encounter changes in extracellular pH (pHe conditions. Upon activation of NADPH oxidase 2 (Nox, neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi. Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET formation (NETosis is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units increased pHi of both activated and resting neutrophils within 10–20 min (Seminaphtharhodafluor dual fluorescence measurements. Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs. In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots and NETosis. Raising pH increased Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative-, and Staphylococcus aureus (Gram-positive-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM increases NETosis. Each Tris molecule can bind 3H+ ions, whereas each bicarbonate HCO3− ion binds 1H+ ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar

A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system.

Science.gov (United States)

Raimondo, Joseph V; Joyce, Bradley; Kay, Louise; Schlagheck, Theresa; Newey, Sarah E; Srinivas, Shankar; Akerman, Colin J

2013-01-01

Within the nervous system, intracellular Cl(-) and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl(-) and pH are often co-regulated, and network activity results in the movement of both Cl(-) and H(+). Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl(-) and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN-a new genetically-encoded ratiometric Cl(-) and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl(-) and H(+) concentrations under a variety of conditions. In addition, we establish the sensor's utility by characterizing activity-dependent ion dynamics in hippocampal neurons.

Label-free silicon nanodots featured ratiometric fluorescent aptasensor for lysosomal imaging and pH measurement.

Science.gov (United States)

Zhang, Yanan; Guo, Shan; Cheng, Shibo; Ji, Xinghu; He, Zhike

2017-08-15

The homeostasis of lysosomal pH is crucial in cell physiology. Developing small fluorescent nanosensors for lysosome imaging and ratiometric measurement of pH is highly demanded yet challenging. Herein, a pH-sensitive fluorescein tagged aptamer AS1411 has been utilized to covalently modify the label-free fluorescent silicon nanodots via a crosslinker for construction of a ratiometric pH biosensor. The established aptasensor exhibits the advantages of ultrasmall size, hypotoxicity, excellent pH reversibility and good photostability, which favors its application in an intracellular environment. Using human breast MCF-7 cancer cells and MCF-10A normal cells as the model, this aptasensor shows cell specificity for cancer cells and displays a wide pH response range of 4.5-8.0 in living cells. The results demonstrate that the pH of MCF-7 cells is 5.1, which is the expected value for acidic organelles. Lysosome imaging and accurate measurement of pH in MCF-7 cells have been successfully conducted based on this nanosensor via fluorescent microscopy and flow cytometry. Copyright © 2017 Elsevier B.V. All rights reserved.

Insecticide resistance and intracellular proteases.

Science.gov (United States)

Wilkins, Richard M

2017-12-01

Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Inferred effects of cloud deposition on forest floor nutrient cycling and microbial properties along a short elevation gradient

International Nuclear Information System (INIS)

Lavoie, M.; Bradley, R.L.

2003-01-01

Higher cloud cover significantly decreases forest floor pH, decrease exchangeable cations, modifies mineral-N speciation and increases physiological stress within microbial communities. - Cloud water deposition often increases with elevation, and it is widely accepted that this cloud water increases acid loading to upland forest ecosystems. A study was undertaken in south-eastern Quebec to determine if a 250 m elevation gradient (i.e. 420-665 m), along a uniform sugar-maple stand on the slope of Mount Orford, corresponded to a pH gradient in the forest floor and to predictable changes in soil nutrient availability and microbial properties. Precipitation data from a nearby study, and a photographic survey, provided presumptive evidence that this elevation gradient corresponded to a strong gradient in cloud water deposition. Forest floor temperature did not differ significantly across elevations. Forest floor moisture content was significantly higher, whereas pH and exchangeable Ca and Mg were significantly lower, at the higher elevations. Average seasonal net nitrification rates, determined by long-term laboratory incubations, did not differ significantly across elevations, whereas average seasonal net ammonification rates were significantly higher at higher elevations. Basal respiration rates and microbial biomass did not differ significantly across elevations, but metabolic quotient was significantly higher at higher elevations indicating possible environmental stress on forest floor microbial communities due to cloud water deposition. Anaerobic N mineralisation rates were significantly higher at higher elevations suggesting that N-limited microbial communities frequently exposed to cloud cover can be important short-term sinks for atmospheric N, thereby contributing to increase the active-N fraction of forest floors. We conclude that, where no significant changes in vegetation or temperature occur, elevation gradients can still be used to understand the spatial

A single pH fluorescent probe for biosensing and imaging of extreme acidity and extreme alkalinity.

Science.gov (United States)

Chao, Jian-Bin; Wang, Hui-Juan; Zhang, Yong-Bin; Li, Zhi-Qing; Liu, Yu-Hong; Huo, Fang-Jun; Yin, Cai-Xia; Shi, Ya-Wei; Wang, Juan-Juan

2017-07-04

A simple tailor-made pH fluorescent probe 2-benzothiazole (N-ethylcarbazole-3-yl) hydrazone (Probe) is facilely synthesized by the condensation reaction of 2-hydrazinobenzothiazole with N-ethylcarbazole-3-formaldehyde, which is a useful fluorescent probe for monitoring extremely acidic and alkaline pH, quantitatively. The pH titrations indicate that Probe displays a remarkable emission enhancement with a pK a of 2.73 and responds linearly to minor pH fluctuations within the extremely acidic range of 2.21-3.30. Interestingly, Probe also exhibits strong pH-dependent characteristics with pK a 11.28 and linear response to extreme-alkalinity range of 10.41-12.43. In addition, Probe shows a large Stokes shift of 84 nm under extremely acidic and alkaline conditions, high selectivity, excellent sensitivity, good water-solubility and fine stability, all of which are favorable for intracellular pH imaging. The probe is further successfully applied to image extremely acidic and alkaline pH values fluctuations in E. coli cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Organelle-targeting surface-enhanced Raman scattering (SERS) nanosensors for subcellular pH sensing.

Science.gov (United States)

Shen, Yanting; Liang, Lijia; Zhang, Shuqin; Huang, Dianshuai; Zhang, Jing; Xu, Shuping; Liang, Chongyang; Xu, Weiqing

2018-01-25

The pH value of subcellular organelles in living cells is a significant parameter in the physiological activities of cells. Its abnormal fluctuations are commonly believed to be associated with cancers and other diseases. Herein, a series of surface-enhanced Raman scattering (SERS) nanosensors with high sensitivity and targeting function was prepared for the quantification and monitoring of pH values in mitochondria, nucleus, and lysosome. The nanosensors were composed of gold nanorods (AuNRs) functionalized with a pH-responsive molecule (4-mercaptopyridine, MPy) and peptides that could specifically deliver the AuNRs to the targeting subcellular organelles. The localization of our prepared nanoprobes in specific organelles was confirmed by super-high resolution fluorescence imaging and bio-transmission electron microscopy (TEM) methods. By the targeting ability, the pH values of the specific organelles can be determined by monitoring the vibrational spectral changes of MPy with different pH values. Compared to the cases of reported lysosome and cytoplasm SERS pH sensors, more accurate pH values of mitochondria and nucleus, which could be two additional intracellular tracers for subcellular microenvironments, were disclosed by this SERS approach, further improving the accuracy of discrimination of related diseases. Our sensitive SERS strategy can also be employed to explore crucial physiological and biological processes that are related to subcellular pH fluctuations.

In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons

KAUST Repository

Mä chler, Philipp; Wyss, Matthias  T.; Elsayed, Maha; Stobart, Jillian; Gutierrez, Robin; von  Faber-Castell, Alexandra; Kaelin, Vincens; Zuend, Marc; San  Martí n, Alejandro; Romero-Gó mez, Ignacio; Baeza-Lehnert, Felipe; Lengacher, Sylvain; Schneider, Bernard  L.; Aebischer, Patrick; Magistretti, Pierre J.; Barros, L.  Felipe; Weber, Bruno

2015-01-01

Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons. © 2016 Elsevier Inc.

In Vivo Evidence for a Lactate Gradient from Astrocytes to Neurons

KAUST Repository

Mächler, Philipp

2015-11-19

Investigating lactate dynamics in brain tissue is challenging, partly because in vivo data at cellular resolution are not available. We monitored lactate in cortical astrocytes and neurons of mice using the genetically encoded FRET sensor Laconic in combination with two-photon microscopy. An intravenous lactate injection rapidly increased the Laconic signal in both astrocytes and neurons, demonstrating high lactate permeability across tissue. The signal increase was significantly smaller in astrocytes, pointing to higher basal lactate levels in these cells, confirmed by a one-point calibration protocol. Trans-acceleration of the monocarboxylate transporter with pyruvate was able to reduce intracellular lactate in astrocytes but not in neurons. Collectively, these data provide in vivo evidence for a lactate gradient from astrocytes to neurons. This gradient is a prerequisite for a carrier-mediated lactate flux from astrocytes to neurons and thus supports the astrocyte-neuron lactate shuttle model, in which astrocyte-derived lactate acts as an energy substrate for neurons. © 2016 Elsevier Inc.

pH is the primary determinant of the bacterial community structure in agricultural soils impacted by polycyclic aromatic hydrocarbon pollution

Science.gov (United States)

Wu, Yucheng; Zeng, Jun; Zhu, Qinghe; Zhang, Zhenfa; Lin, Xiangui

2017-01-01

Acidification and pollution are two major threats to agricultural ecosystems; however, microbial community responses to co-existed soil acidification and pollution remain less explored. In this study, arable soils of broad pH (4.26-8.43) and polycyclic aromatic hydrocarbon (PAH) gradients (0.18-20.68 mg kg-1) were collected from vegetable farmlands. Bacterial community characteristics including abundance, diversity and composition were revealed by quantitative PCR and high-throughput sequencing. The bacterial 16S rRNA gene copies significantly correlated with soil carbon and nitrogen contents, suggesting the control of nutrients accessibility on bacterial abundance. The bacterial diversity was strongly related to soil pH, with higher diversity in neutral samples and lower in acidic samples. Soil pH was also identified by an ordination analysis as important factor shaping bacterial community composition. The relative abundances of some dominant phyla varied along the pH gradient, and the enrichment of a few phylotypes suggested their adaptation to low pH condition. In contrast, at the current pollution level, PAH showed marginal effects on soil bacterial community. Overall, these findings suggest pH was the primary determinant of bacterial community in these arable soils, indicative of a more substantial influence of acidification than PAH pollution on bacteria driven ecological processes.

Magnetogenetic control of protein gradients inside living cells with high spatial and temporal resolution.

Science.gov (United States)

Etoc, Fred; Vicario, Chiara; Lisse, Domenik; Siaugue, Jean-Michel; Piehler, Jacob; Coppey, Mathieu; Dahan, Maxime

2015-05-13

Tools for controlling the spatial organization of proteins are a major prerequisite for deciphering mechanisms governing the dynamic architecture of living cells. Here, we have developed a generic approach for inducing and maintaining protein gradients inside living cells by means of biofunctionalized magnetic nanoparticles (MNPs). For this purpose, we tailored the size and surface properties of MNPs in order to ensure unhindered mobility in the cytosol. These MNPs with a core diameter below 50 nm could be rapidly relocalized in living cells by exploiting biased diffusion at weak magnetic forces in the femto-Newton range. In combination with MNP surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, we here present a comprehensive technology for controlling intracellular protein gradients with a temporal resolution of a few tens of seconds.

A genetic variant of the sperm-specific SLO3 K+ channel has altered pH and Ca2+ sensitivities.

Science.gov (United States)

Geng, Yanyan; Ferreira, Juan J; Dzikunu, Victor; Butler, Alice; Lybaert, Pascale; Yuan, Peng; Magleby, Karl L; Salkoff, Lawrence; Santi, Celia M

2017-05-26

To fertilize an oocyte, sperm must first undergo capacitation in which the sperm plasma membrane becomes hyperpolarized via activation of potassium (K + ) channels and resultant K + efflux. Sperm-specific SLO3 K + channels are responsible for these membrane potential changes critical for fertilization in mouse sperm, and they are only sensitive to pH i However, in human sperm, the major K + conductance is both Ca 2+ - and pH i -sensitive. It has been debated whether Ca 2+ -sensitive SLO1 channels substitute for human SLO3 (hSLO3) in human sperm or whether human SLO3 channels have acquired Ca 2+ sensitivity. Here we show that hSLO3 is rapidly evolving and reveal a natural structural variant with enhanced apparent Ca 2+ and pH sensitivities. This variant allele (C382R) alters an amino acid side chain at a principal interface between the intramembrane-gated pore and the cytoplasmic gating ring of the channel. Because the gating ring contains sensors to intracellular factors such as pH and Ca 2+ , the effectiveness of transduction between the gating ring and the pore domain appears to be enhanced. Our results suggest that sperm-specific genes can evolve rapidly and that natural genetic variation may have led to a SLO3 variant that differs from wild type in both pH and intracellular Ca 2+ sensitivities. Whether this physiological variation confers differences in fertility among males remains to be established. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system

Science.gov (United States)

Raimondo, Joseph V.; Joyce, Bradley; Kay, Louise; Schlagheck, Theresa; Newey, Sarah E.; Srinivas, Shankar; Akerman, Colin J.

2013-01-01

Within the nervous system, intracellular Cl− and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission, and network excitability. Cl− and pH are often co-regulated, and network activity results in the movement of both Cl− and H+. Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl− and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN—a new genetically-encoded ratiometric Cl− and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl− and H+ concentrations under a variety of conditions. In addition, we establish the sensor's utility by characterizing activity-dependent ion dynamics in hippocampal neurons. PMID:24312004

A genetically-encoded chloride and pH sensor for dissociating ion dynamics in the nervous system

Directory of Open Access Journals (Sweden)

Joseph Valentino Raimondo

2013-11-01

Full Text Available Within the nervous system, intracellular Cl- and pH regulate fundamental processes including cell proliferation, metabolism, synaptic transmission and network excitability. Cl- and pH are often co-regulated, and network activity results in the movement of both Cl- and H+. Tools to accurately measure these ions are crucial for understanding their role under physiological and pathological conditions. Although genetically-encoded Cl- and pH sensors have been described previously, these either lack ion specificity or are unsuitable for neuronal use. Here we present ClopHensorN - a new genetically-encoded ratiometric Cl- and pH sensor that is optimized for the nervous system. We demonstrate the ability of ClopHensorN to dissociate and simultaneously quantify Cl- and H+ concentrations under a variety of conditions. In addition, we establish the sensor’s utility by characterizing activity-dependent ion dynamics in hippocampal neurons.

Ca2+-associated triphasic pH changes in mitochondria during brown adipocyte activation.

Science.gov (United States)

Hou, Yanyan; Kitaguchi, Tetsuya; Kriszt, Rókus; Tseng, Yu-Hua; Raghunath, Michael; Suzuki, Madoka

2017-08-01

Brown adipocytes (BAs) are endowed with a high metabolic capacity for energy expenditure due to their high mitochondria content. While mitochondrial pH is dynamically regulated in response to stimulation and, in return, affects various metabolic processes, how mitochondrial pH is regulated during adrenergic stimulation-induced thermogenesis is unknown. We aimed to reveal the spatial and temporal dynamics of mitochondrial pH in stimulated BAs and the mechanisms behind the dynamic pH changes. A mitochondrial targeted pH-sensitive protein, mito-pHluorin, was constructed and transfected to BAs. Transfected BAs were stimulated by an adrenergic agonist, isoproterenol. The pH changes in mitochondria were characterized by dual-color imaging with indicators that monitor mitochondrial membrane potential and heat production. The mechanisms of pH changes were studied by examining the involvement of electron transport chain (ETC) activity and Ca 2+ profiles in mitochondria and the intracellular Ca 2+ store, the endoplasmic reticulum (ER). A triphasic mitochondrial pH change in BAs upon adrenergic stimulation was revealed. In comparison to a thermosensitive dye, we reveal that phases 1 and 2 of the pH increase precede thermogenesis, while phase 3, characterized by a pH decrease, occurs during thermogenesis. The mechanism of pH increase is partially related to ETC. In addition, the pH increase occurs concurrently with an increase in mitochondrial Ca 2+ . This Ca 2+ increase is contributed to by an influx from the ER, and it is further involved in mitochondrial pH regulation. We demonstrate that an increase in mitochondrial pH is implicated as an early event in adrenergically stimulated BAs. We further suggest that this pH increase may play a role in the potentiation of thermogenesis.

Protein Complexation and pH Dependent Release Using Boronic Acid Containing PEG-Polypeptide Copolymers.

Science.gov (United States)

Negri, Graciela E; Deming, Timothy J

2017-01-01

New poly(L-lysine)-b-poly(ethylene glycol) copolypeptides have been prepared, where the side-chain amine groups of lysine residues are modified to contain ortho-amine substituted phenylboronic acid, i.e., Wulff-type phenylboronic acid (WBA), groups to improve their pH responsive, carbohydrate binding properties. These block copolymers form nanoscale complexes with glycosylated proteins that are stable at physiological pH, yet dissociate and release the glycoproteins under acidic conditions, similar to those found in endosomal and lysosomal compartments within cells. These results suggest that WBA modified polypeptide copolymers are promising for further development as degradable carriers for intracellular protein delivery. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Monitoring the corrosion process of Al alloys through pH induced fluorescence

International Nuclear Information System (INIS)

Pidaparti, R M; Neblett, E B; Miller, S A; Alvarez, J C

2008-01-01

A sensing and monitoring set-up based on electrochemical pH induced fluorescence to systematically control the electrochemical corrosion process has been developed for possible applications in the field of localized corrosion. The sensing and monitoring concept is based on exposing the corroding metal surface to solutions that contain selected redox chemicals which will react in local regions where anodic or cathodic polarizations occur. Redox couples that produce or consume protons in their electrochemical reactions were used so that local pH gradients can indicate electrochemical activity by inducing fluorescence in dyes. This approach has been applied to study the corrosion initiation in aircraft aluminum metal 2024-T3 in a controlled electrochemical cell. Preliminary results obtained suggest that monitoring of localized corrosion based on pH can be achieved for field applications

Effect of ATP on intracellular pH in pancreatic ducts involves P2X7 receptors

DEFF Research Database (Denmark)

Henriksen, Katerine L; Novak, Ivana

2003-01-01

which P2 receptors might be involved. Ducts were obtained from rat pancreas, and the pH sensitive fluorophore BCECF was used to measure pHi and recovery rates from cellular acidosis induced by ammonium pre-pulses. In order to reveal Na+/H+ exchange, Cl-/HCO3- exchange or a Na+-HCO3- cotransport...

Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment.

Science.gov (United States)

Xia, J. H.; Roberts, JKM.

1996-05-01

We tested the hypothesis that H+ extrusion contributes to cytoplasmic pH regulation and tolerance of anoxia in maize (Zea mays) root tips. We studied root tips of whole seedlings that were acclimated to a low-oxygen environment by pretreatment in 3% (v/v) O2. Acclimated root tips characteristically regulate cytoplasmic pH near neutrality and survive prolonged anoxia, whereas nonacclimated tips undergo severe cytoplasmic acidosis and die much more quickly. We show that the plasma membrane H+-ATPase can operate under anoxia and that net H+ extrusion increases when cytoplasmic pH falls. However, at an external pH near 6.0, H+ extrusion contributes little to cytoplasmic pH regulation. At more acidic external pH values, net H+ flux into root tips increases dramatically, leading to a decrease in cytoplasmic pH and reduced tolerance of anoxia. We present evidence that, under these conditions, H+ pumps are activated to partly offset acidosis due to H+ influx and, thereby, contribute to cytoplasmic pH regulation and tolerance of anoxia. The regulation of H+ extrusion under anoxia is discussed with respect to the acclimation response and mechanisms of intracellular pH regulation in aerobic plant cells.

Transport in Halobacterium Halobium: Light-Induced Cation-Gradients, Amino Acid Transport Kinetics, and Properties of Transport Carriers

Science.gov (United States)

Lanyi, Janos K.

1977-01-01

Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na(+). Measurements of Na-22 flux, exterior pH change, and membrane potential, Delta(psi) (with the dye 3,3'-dipentyloxadicarbocyanine) indicate that the means of Na(+) transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H(+)/Na(++ greater than 1). The resulting large chemical gradient for Na(+) (outside much greater than inside), as well as the membrane potential, will drive the transport of 18 amino acids. The I9th, glutamate, is unique in that its accumulation is indifferent to Delta(psi): this amino acid is transported only when a chemical gradient for Na(+) is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H+() collapses within 1 min, while the large Na(+) gradient and glutamate transporting activity persists for 10- 15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na(+), arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V(sub max) and K(sub m) comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na(+), in an electrically neutral fashion, so that only the chemical component of the Na(+) gradient is a driving force.

Fabrication of CTP/HAp novel gradient composite bioceramics

Institute of Scientific and Technical Information of China (English)

无

2007-01-01

Calcium-titanium-phosphate(CaTi4(PO4)6, CTP)/hydroxyapatite(HAp) is a kind of novel gradient composite bioceramics,which has excellent biocompatibility and bioactivity. CTP ceramic film was synthesized one-step on the surface of titanium using micro-arc oxidation(MAO). The CTP/HAp composite bioceramics were prepared by soaking CTP film in HAp inducing solution for several days. XRD, SEM and EDX were used to characterize the bio-ceramic films phase and composition, morphology and component. The influence of electrolyte molar ratio of Ca to P and the current density to the synthesis of film was studied, and the optimized value of parameters above were 1/6 and 15A/dm2. The parameters of HAp inducing solution, such as component and pH value were also studied and the best pH value which is adjusted by NaOH is 6.4.

Intracellular pH in human brain measured by P-31 MR spectroscopy during changes in arterial CO/sub 2/ tension

International Nuclear Information System (INIS)

Jensen, K.E.; Thomsen, C.; Henriksen, O.

1987-01-01

Six healthy volunteers were examined. A 1.5-T Siemens whole-body scanner was used. A three-turn solenoid surface coil was used. Sixty-four acquisitions with a repetition time of 6 seconds were recorded. The subjects hyperventilated and inhaled air with 5% and 7% CO/sub 2/. The breathing of air with 5% CO/sub 2/ resulted in an arterial blood tension of 40 mm Hg, and no changes in pH could be detected. Breathing of air with 7% CO/sub 2/ resulted in arterial tension of 55 mm Hg and gave a decrease of 0.1 pH unit. The spectra after 15 minutes of hyperventilation showed an increase of 0.1 pH unit

Potential role of sodium-proton exchangers in the low concentration arsenic trioxide-increased intracellular pH and cell proliferation.

Directory of Open Access Journals (Sweden)

Carmen Aravena

Full Text Available Arsenic main inorganic compound is arsenic trioxide (ATO presented in solution mainly as arsenite. ATO increases intracellular pH (pHi, cell proliferation and tumor growth. Sodium-proton exchangers (NHEs modulate the pHi, with NHE1 playing significant roles. Whether ATO-increased cell proliferation results from altered NHEs expression and activity is unknown. We hypothesize that ATO increases cell proliferation by altering pHi due to increased NHEs-like transport activity. Madin-Darby canine kidney (MDCK cells grown in 5 mmol/L D-glucose-containing DMEM were exposed to ATO (0.05, 0.5 or 5 µmol/L, 0-48 hours in the absence or presence of 5-N,N-hexamethylene amiloride (HMA, 5-100 µmol/L, NHEs inhibitor, PD-98059 (30 µmol/L, MAPK1/2 inhibitor, Gö6976 (10 µmol/L, PKCα, βI and μ inhibitor, or Schering 28080 (10 µmol/L, H(+/K(+ATPase inhibitor plus concanamycin (0.1 µmol/L, V type ATPases inhibitor. Incorporation of [(3H]thymidine was used to estimate cell proliferation, and counting cells with a hemocytometer to determine the cell number. The pHi was measured by fluorometry in 2,7-bicarboxyethyl-5,6-carboxyfluorescein loaded cells. The Na(+-dependent HMA-sensitive NHEs-like mediated proton transport kinetics, NHE1 protein abundance in the total, cytoplasm and plasma membrane protein fractions, and phosphorylated and total p42/44 mitogen-activated protein kinases (p42/44(mapk were also determined. Lowest ATO (0.05 µmol/L, ~0.01 ppm used in this study increased cell proliferation, pHi, NHEs-like transport and plasma membrane NHE1 protein abundance, effects blocked by HMA, PD-98059 or Gö6976. Cell-buffering capacity did not change by ATO. The results show that a low ATO concentration increases MDCK cells proliferation by NHEs (probably NHE1-like transport dependent-increased pHi requiring p42/44(mapk and PKCα, βI and/or μ activity. This finding could be crucial in diseases where uncontrolled cell growth occurs, such as tumor growth, and

Characterization of Growing Soil Bacterial Communities across a pH gradient Using H218O DNA-Stable Isotope Probing

Science.gov (United States)

Welty-Bernard, A. T.; Schwartz, E.

2014-12-01

Recent studies have established consistent relationships between pH and bacterial diversity and community structure in soils from site-specific to landscape scales. However, these studies rely on DNA or PLFA extraction techniques from bulk soils that encompass metabolically active and inactive, or dormant, communities, and loose DNA. Dormant cells may comprise up to 80% of total live cells. If dormant cells dominate a particular environment, it is possible that previous interpretations of the soil variables assumed to drive communities could be profoundly affected. We used H218O stable isotope probing and bar-coded illumina sequencing of 16S rRNA genes to monitor the response of actively growing communities to changes in soil pH in a soil microcosm over 14 days. This substrate-independent approach has several advantages over 13C or 15N-labelled molecules in that all growing bacteria should be able to make use of water, allowing characterization of whole communities. We hypothesized that Acidobacteria would increasingly dominate the growing community and that Actinobacteria and Bacteroidetes would decline, given previously established responses by these taxa to soil pH. Instead, we observed the reverse. Actinobacteria abundance increased three-fold from 26 to 76% of the overall community as soil pH fell from pH 5.6 to pH 4.6. Shifts in community structure and decreases in diversity with declining soil pH were essentially driven by two families, Streptomyceaca and Microbacteracea, which collectively increased from 2 to 40% of the entire community. In contrast, Acidobacteria as a whole declined although numbers of subdivision 1 remained stable across all soil pH levels. We suggest that the brief incubation period in this SIP study selected for growth of acid-tolerant Actinobacteria over Acidobacteria. Taxa within Actinomycetales have been readily cultured over short time frames, suggesting rapid growth patterns. Conversely, taxa within Acidobacteria have been

Effects of calcium, inorganic phosphate, and pH on isometric force in single skinned cardiomyocytes from donor and failing human hearts

NARCIS (Netherlands)

van der Velden, J.; Klein, L. J.; Zaremba, R.; Boontje, N. M.; Huybregts, M. A.; Stooker, W.; Eijsman, L.; de Jong, J. W.; Visser, C. A.; Visser, F. C.; Stienen, G. J.

2001-01-01

During ischemia, the intracellular calcium and inorganic phosphate (P(i)) concentrations rise and pH falls. We investigated the effects of these changes on force development in donor and failing human hearts to determine if altered contractile protein composition during heart failure changes the

Nanoparticles for intracellular-targeted drug delivery

International Nuclear Information System (INIS)

Paulo, Cristiana S O; Pires das Neves, Ricardo; Ferreira, Lino S

2011-01-01

Nanoparticles (NPs) are very promising for the intracellular delivery of anticancer and immunomodulatory drugs, stem cell differentiation biomolecules and cell activity modulators. Although initial studies in the area of intracellular drug delivery have been performed in the delivery of DNA, there is an increasing interest in the use of other molecules to modulate cell activity. Herein, we review the latest advances in the intracellular-targeted delivery of short interference RNA, proteins and small molecules using NPs. In most cases, the drugs act at different cellular organelles and therefore the drug-containing NPs should be directed to precise locations within the cell. This will lead to the desired magnitude and duration of the drug effects. The spatial control in the intracellular delivery might open new avenues to modulate cell activity while avoiding side-effects.

Patterns of development of unspecific reaction of cells and modification of chemical protection

International Nuclear Information System (INIS)

Veksler, A.M.; Korystov, Yu.N.; Kublik, L.N.; Ehjdus, L.Kh.

1980-01-01

A study was made of a correlation between radioprotective efficiency of different chemical agents (weak electrolytes) and conditions of treatment. It was demonstrated that the pattern of changes in the protection efficiency, with modification thereof, is similar to that of the development of unspecific reaction and determined by the intracellular concentration of the chemical agents, which, in turn, is function of physicochemical parameters of the substance and pH gradient between cell and medium. With similar intracellular concentration, caffeine-benzoate, thioglicolic acid and caffeine proved to be equally effective, while the protective effect of cysteamine was appreciably higher

Dual-Emitting Fluorescent Metal-Organic Framework Nanocomposites as a Broad-Range pH Sensor for Fluorescence Imaging.

Science.gov (United States)

Chen, Haiyong; Wang, Jing; Shan, Duoliang; Chen, Jing; Zhang, Shouting; Lu, Xiaoquan

2018-05-15

pH plays an important role in understanding physiological/pathologic processes, and abnormal pH is a symbol of many common diseases such as cancer, stroke, and Alzheimer's disease. In this work, an effective dual-emission fluorescent metal-organic framework nanocomposite probe (denoted as RB-PCN) has been constructed for sensitive and broad-range detection of pH. RB-PCN was prepared by encapsulating the DBI-PEG-NH 2 -functionalized Fe 3 O 4 into Zr-MOFs and then further reacting it with rhodamine B isothiocyanates (RBITC). In RB-PCN, RBITC is capable of sensing changes in pH in acidic solutions. Zr-MOFs not only enrich the target analyte but also exhibit a fluorescence response to pH changes in alkaline solutions. Based on the above structural and compositional features, RB-PCN could detect a wide range of pH changes. Importantly, such a nanoprobe could "see" the intracellular pH changes by fluorescence confocal imaging as well as "measure" the wider range of pH in actual samples by fluorescence spectroscopy. To the best of our knowledge, this is the first time a MOF-based dual-emitting fluorescent nanoprobe has been used for a wide range of pH detection.

Characterization of PEBBLEs as a Tool for Real-Time Measurement of Dictyostelium discoideum Endosomal pH

Directory of Open Access Journals (Sweden)

Everett Moding

2009-01-01

Full Text Available The measurement of intracellular ion concentration change is important for understanding the cellular mechanisms for communication. Recently developed nanosensors, (Photonic Explorers for Biomedical use with Biologically Localized Embedding PEBBLEs, have a number of advantages for measuring ions in cells over established methods using microelectrodes, unbound fluorescent dyes, or NMR. PEBBLE sensors have been shown to work in principle for measuring dynamic ion changes, but few in vivo applications have been demonstrated. We modified the protocol for the fabrication of pH sensing PEBBLEs and developed a protocol for the utilization of these sensors for the monitoring of dynamic pH changes in the endosomes of slime mold Dictyostelium discoideum (D. discoideum. Oregon Green 514-CdSe Quantum Dot PEBBLEs were used to measure real-time pH inside D. discoideum endosomes during cAMP stimulation. Endosomal pH was shown to decrease during cAMP signaling, demonstrating a movement of protons into the endosomes of D. discoideum amoebae.

Dual-lifetime referencing (DLR: a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts

Directory of Open Access Journals (Sweden)

Boniello Caterina

2012-03-01

Full Text Available Abstract Background Industrial-scale biocatalytic synthesis of fine chemicals occurs preferentially as continuous processes employing immobilized enzymes on insoluble porous carriers. Diffusional effects in these systems often create substrate and product concentration gradients between bulk liquid and the carrier. Moreover, some widely-used biotransformation processes induce changes in proton concentration. Unlike the bulk pH, which is usually controlled at a suitable value, the intraparticle pH of immobilized enzymes may deviate significantly from its activity and stability optima. The magnitude of the resulting pH gradient depends on the ratio of characteristic times for enzymatic reaction and on mass transfer (the latter is strongly influenced by geometrical features of the porous carrier. Design and selection of optimally performing enzyme immobilizates would therefore benefit largely from experimental studies of the intraparticle pH environment. Here, a simple and non-invasive method based on dual-lifetime referencing (DLR for pH determination in immobilized enzymes is introduced. The technique is applicable to other systems in which particles are kept in suspension by agitation. Results The DLR method employs fluorescein as pH-sensitive luminophore and Ru(II tris(4,7-diphenyl-1,10-phenantroline, abbreviated Ru(dpp, as the reference luminophore. Luminescence intensities of the two luminophores are converted into an overall phase shift suitable for pH determination in the range 5.0-8.0. Sepabeads EC-EP were labeled by physically incorporating lipophilic variants of the two luminophores into their polymeric matrix. These beads were employed as carriers for immobilization of cephalosporin C amidase (a model enzyme of industrial relevance. The luminophores did not interfere with the enzyme immobilization characteristics. Analytical intraparticle pH determination was optimized for sensitivity, reproducibility and signal stability under

Biodegradable "Smart" Polyphosphazenes with Intrinsic Multifunctionality as Intracellular Protein Delivery Vehicles.

Science.gov (United States)

Martinez, Andre P; Qamar, Bareera; Fuerst, Thomas R; Muro, Silvia; Andrianov, Alexander K

2017-06-12

A series of biodegradable drug delivery polymers with intrinsic multifunctionality have been designed and synthesized utilizing a polyphosphazene macromolecular engineering approach. Novel water-soluble polymers, which contain carboxylic acid and pyrrolidone moieties attached to an inorganic phosphorus-nitrogen backbone, were characterized by a suite of physicochemical methods to confirm their structure, composition, and molecular sizes. All synthesized polyphosphazenes displayed composition-dependent hydrolytic degradability in aqueous solutions at neutral pH. Their formulations were stable at lower temperatures, potentially indicating adequate shelf life, but were characterized by accelerated degradation kinetics at elevated temperatures, including 37 °C. It was found that synthesized polyphosphazenes are capable of environmentally triggered self-assembly to produce nanoparticles with narrow polydispersity in the size range of 150-700 nm. Protein loading capacity of copolymers has been validated via their ability to noncovalently bind avidin without altering biological functionality. Acid-induced membrane-disruptive activity of polyphosphazenes has been established with an onset corresponding to the endosomal pH range and being dependent on polymer composition. The synthesized polyphosphazenes facilitated cell-surface interactions followed by time-dependent, vesicular-mediated, and saturable internalization of a model protein cargo into cancer cells, demonstrating the potential for intracellular delivery.

pH is the primary determinant of the bacterial community structure in agricultural soils impacted by polycyclic aromatic hydrocarbon pollution

Science.gov (United States)

Wu, Yucheng; Zeng, Jun; Zhu, Qinghe; Zhang, Zhenfa; Lin, Xiangui

2017-01-01

Acidification and pollution are two major threats to agricultural ecosystems; however, microbial community responses to co-existed soil acidification and pollution remain less explored. In this study, arable soils of broad pH (4.26–8.43) and polycyclic aromatic hydrocarbon (PAH) gradients (0.18–20.68 mg kg−1) were collected from vegetable farmlands. Bacterial community characteristics including abundance, diversity and composition were revealed by quantitative PCR and high-throughput sequencing. The bacterial 16S rRNA gene copies significantly correlated with soil carbon and nitrogen contents, suggesting the control of nutrients accessibility on bacterial abundance. The bacterial diversity was strongly related to soil pH, with higher diversity in neutral samples and lower in acidic samples. Soil pH was also identified by an ordination analysis as important factor shaping bacterial community composition. The relative abundances of some dominant phyla varied along the pH gradient, and the enrichment of a few phylotypes suggested their adaptation to low pH condition. In contrast, at the current pollution level, PAH showed marginal effects on soil bacterial community. Overall, these findings suggest pH was the primary determinant of bacterial community in these arable soils, indicative of a more substantial influence of acidification than PAH pollution on bacteria driven ecological processes. PMID:28051171

Effects of anatomy and diet on gastrointestinal pH in rodents.

Science.gov (United States)

Kohl, Kevin D; Stengel, Ashley; Samuni-Blank, Michal; Dearing, M Denise

2013-04-01

The pH of the gastrointestinal tract can have profound influences on digestive processes. Rodents exhibit wide variation in both stomach morphology and dietary strategies, both of which may influence gut pH. Various rodent species have evolved bilocular (or semi-segmented) stomachs that may allow for more microbial growth compared to unilocular (single-chambered) stomachs. Additionally, herbivory has evolved multiple times in rodents. The high dietary fiber typical of an herbivorous diet is known to induce secretion of bicarbonate in the gut. We predicted that stomach segmentation might facilitate the separation of contents in the proximal chamber from that of the gastric stomach, facilitating a chemical environment suitable to microbial growth. To investigate the effect of stomach anatomy and diet on gut pH, several species of rodent with varying stomach morphology were fed either a high or low-fiber diet for 7 days, and pH of the proximal stomach, gastric stomach, small intestine, and cecum were measured. We discovered that rodents with bilocular stomach anatomy maintained a larger pH gradient between the proximal and gastric stomach compartments, and were able to achieve a lower absolute gastric pH compared to those with unilocular stomachs. Dietary fiber increased the pH of the small intestine, but not in any other gut regions. The stomach pH data supports the century old hypothesis that bilocular stomach anatomy creates an environment in the proximal stomach that is suitable for microbial growth. Additionally, the alkaline small intestinal pH on a high fiber diet may enhance digestion. Copyright © 2013 Wiley Periodicals, Inc.

The alteration of intracellular enzymes. III. The effect of temperature on the kinetics of altered and unaltered yeast catalase.

Science.gov (United States)

FRASER, M J; KAPLAN, J G

1955-03-20

the case of catalase, of desorbing the enzyme from the interface into its rolled-up, soluble, highly specific configuration. While the interfacial hypothesis has successfully withstood this experimental attack, the present data do not provide its unequivocal proof, since they are consistent with any hypothesis of alteration in which the unaltered, intracellular enzyme is in a relatively disordered state by comparison to the altered enzyme. While evidence of an interfacial process in enzyme alteration has been adduced previously, critical proof of the interfacial hypothesis awaits creation of a model system, in which most of the aspects of intracellular alteration can be reproduced. 6. Certain of the changes in kinetic properties following alteration of the intracellular enzyme, such as increased activity and the modified energies and entropies of activation of both enzyme-substrate system and heat destruction of the catalase itself, might be explained by a decrease (two orders of magnitude) in the effective hydrogen ion concentration, allowing the intracellular enzyme to be brought to the same pH as the extracellular medium. If such a pH change does, in fact, occur, it is necessary to invoke the interfacial hypothesis to explain why the unaltered, intracellular enzyme is in equilibrium with a medium whose pH is approximately 2 units lower than that of the cytoplasm itself. 7. It is concluded that kinetic data of this kind may be used to shed light on the structure of a soluble, cytoplasmic enzyme, not attached to any of the formed elements within the cell, yet organized within it in a condition of relatively low structural specificity; further, that information obtained exclusively from a study of the kinetics of the extracted or crystalline enzymes may not, in the case of this enzyme, at least, be extrapolated to the same enzyme within the intact cell.

Efficient intracellular delivery and improved biocompatibility of colloidal silver nanoparticles towards intracellular SERS immuno-sensing.

Science.gov (United States)

Bhardwaj, Vinay; Srinivasan, Supriya; McGoron, Anthony J

2015-06-21

High throughput intracellular delivery strategies, electroporation, passive and TATHA2 facilitated diffusion of colloidal silver nanoparticles (AgNPs) are investigated for cellular toxicity and uptake using state-of-art analytical techniques. The TATHA2 facilitated approach efficiently delivered high payload with no toxicity, pre-requisites for intracellular applications of plasmonic metal nanoparticles (PMNPs) in sensing and therapeutics.

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

Science.gov (United States)

Jiang, Fei; Kongsaeree, Puapong; Schilke, Karl; Lajoie, Curtis; Kelly, Christine

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris αMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris αMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris ctMnP1-1 were determined to be pH 6 and 30 °C, respectively. P. pastoris αMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris αMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4-7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.

Perturbation Analysis of Calcium, Alkalinity and Secretion during Growth of Lily Pollen Tubes.

Science.gov (United States)

Winship, Lawrence J; Rounds, Caleb; Hepler, Peter K

2016-12-30

Pollen tubes grow by spatially and temporally regulated expansion of new material secreted into the cell wall at the tip of the tube. A complex web of interactions among cellular components, ions and small molecule provides dynamic control of localized expansion and secretion. Cross-correlation studies on oscillating lily ( Lilium formosanum Wallace) pollen tubes showed that an increase in intracellular calcium follows an increase in growth, whereas the increase in the alkaline band and in secretion both anticipate the increase in growth rate. Calcium, as a follower, is unlikely to be a stimulator of growth, whereas the alkaline band, as a leader, may be an activator. To gain further insight herein we reversibly inhibited growth with potassium cyanide (KCN) and followed the re-establishment of calcium, pH and secretion patterns as growth resumed. While KCN markedly slows growth and causes the associated gradients of calcium and pH to sharply decline, its removal allows growth and vital processes to fully recover. The calcium gradient reappears before growth restarts; however, it is preceded by both the alkaline band and secretion, in which the alkaline band is slightly advanced over secretion. Thus the pH gradient, rather than the tip-focused calcium gradient, may regulate pollen tube growth.

Perturbation Analysis of Calcium, Alkalinity and Secretion during Growth of Lily Pollen Tubes

Directory of Open Access Journals (Sweden)

Lawrence J. Winship

2016-12-01

Full Text Available Pollen tubes grow by spatially and temporally regulated expansion of new material secreted into the cell wall at the tip of the tube. A complex web of interactions among cellular components, ions and small molecule provides dynamic control of localized expansion and secretion. Cross-correlation studies on oscillating lily (Lilium formosanum Wallace pollen tubes showed that an increase in intracellular calcium follows an increase in growth, whereas the increase in the alkaline band and in secretion both anticipate the increase in growth rate. Calcium, as a follower, is unlikely to be a stimulator of growth, whereas the alkaline band, as a leader, may be an activator. To gain further insight herein we reversibly inhibited growth with potassium cyanide (KCN and followed the re-establishment of calcium, pH and secretion patterns as growth resumed. While KCN markedly slows growth and causes the associated gradients of calcium and pH to sharply decline, its removal allows growth and vital processes to fully recover. The calcium gradient reappears before growth restarts; however, it is preceded by both the alkaline band and secretion, in which the alkaline band is slightly advanced over secretion. Thus the pH gradient, rather than the tip-focused calcium gradient, may regulate pollen tube growth.

Na+, HCO3--cotransporter NBCn1 increases pHi gradients, filopodia, and migration of smooth muscle cells and promotes arterial remodelling.

Science.gov (United States)

Boedtkjer, Ebbe; Bentzon, Jacob F; Dam, Vibeke S; Aalkjaer, Christian

2016-08-01

Arterial remodelling can cause luminal narrowing and obstruct blood flow. We tested the hypothesis that cellular acid-base transport facilitates proliferation and migration of vascular smooth muscle cells (VSMCs) and enhances remodelling of conduit arteries. [Formula: see text]-cotransport via NBCn1 (Slc4a7) mediates net acid extrusion and controls steady-state intracellular pH (pHi) in VSMCs of mouse carotid arteries and primary aortic explants. Carotid arteries undergo hypertrophic inward remodelling in response to partial or complete ligation in vivo, but the increase in media area and thickness and reduction in lumen diameter are attenuated in arteries from NBCn1 knock-out compared with wild-type mice. With [Formula: see text] present, gradients for pHi (∼0.2 units magnitude) exist along the axis of VSMC migration in primary explants from wild-type but not NBCn1 knock-out mice. Knock-out or pharmacological inhibition of NBCn1 also reduces filopodia and lowers initial rates of VSMC migration after scratch-wound infliction. Interventions to reduce H(+)-buffer mobility (omission of [Formula: see text] or inhibition of carbonic anhydrases) re-establish axial pHi gradients, filopodia, and migration rates in explants from NBCn1 knock-out mice. The omission of [Formula: see text] also lowers global pHi and inhibits proliferation in primary explants. Under physiological conditions (i.e. with [Formula: see text] present), NBCn1-mediated [Formula: see text] uptake raises VSMC pHi and promotes filopodia, VSMC migration, and hypertrophic inward remodelling. We propose that axial pHi gradients enhance VSMC migration whereas global acidification inhibits VSMC proliferation and media hypertrophy after carotid artery ligation. These findings support a key role of acid-base transport, particularly via NBCn1, for development of occlusive artery disease. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please

An intramolecular charge transfer process based fluorescent probe for monitoring subtle pH fluctuation in living cells.

Science.gov (United States)

Sun, Mingtai; Du, Libo; Yu, Huan; Zhang, Kui; Liu, Yang; Wang, Suhua

2017-01-01

It is crucial to monitor intracellular pH values and their fluctuation since the organelles of cells have different pH distribution. Herein we construct a new small molecule fluorescent probe HBT-O for monitoring the subtle pH values within the scope of neutral to acid in living cells. The probe exhibited good water solubility, a marked turquoise to olivine emission color change in response to pH, and tremendous fluorescence hypochromatic shift of ∼50nm (1718cm -1 ) as well as the increased fluorescence intensity when the pH value changed from neutral to acid. Thus, the probe HBT-O can distinguish the subtle changes in the range of normal pH values from neutral to acid with significant fluorescence changes. These properties can be attributed to the intramolecular charge transfer (ICT) process of the probe upon protonation in buffer solutions at varied pH values. Moreover, the probe was reversible and nearly non-toxic for living cells. Then the probe was successfully used to detect pH fluctuation in living cells by exhibiting different fluorescence colors and intensity. These findings demonstrate that the probe will find useful applications in biology and biomedical research. Copyright © 2016 Elsevier B.V. All rights reserved.

pH-Dependent Toxicity of High Aspect Ratio ZnO Nanowires in Macrophages Due to Intracellular Dissolution

KAUST Repository

H. Müller, Karin

2010-11-23

High-aspect ratio ZnO nanowires have become one of the most promising products in the nanosciences within the past few years with a multitude of applications at the interface of optics and electronics. The interaction of zinc with cells and organisms is complex, with both deficiency and excess causing severe effects. The emerging significance of zinc for many cellular processes makes it imperative to investigate the biological safety of ZnO nanowires in order to guarantee their safe economic exploitation. In this study, ZnO nanowires were found to be toxic to human monocyte macrophages (HMMs) at similar concentrations as ZnCl2. Confocal microscopy on live cells confirmed a rise in intracellular Zn2+ concentrations prior to cell death. In vitro, ZnO nanowires dissolved very rapidly in a simulated body fluid of lysosomal pH, whereas they were comparatively stable at extracellular pH. Bright-field transmission electron microscopy (TEM) showed a rapid macrophage uptake of ZnO nanowire aggregates by phagocytosis. Nanowire dissolution occurred within membrane-bound compartments, triggered by the acidic pH of the lysosomes. ZnO nanowire dissolution was confirmed by scanning electron microscopy/energy-dispersive X-ray spectrometry. Deposition of electron-dense material throughout the ZnO nanowire structures observed by TEM could indicate adsorption of cellular components onto the wires or localized zinc-induced protein precipitation. Our study demonstrates that ZnO nanowire toxicity in HMMs is due to pH-triggered, intracellular release of ionic Zn2+ rather than the high-aspect nature of the wires. Cell death had features of necrosis as well as apoptosis, with mitochondria displaying severe structural changes. The implications of these findings for the application of ZnO nanowires are discussed. © 2010 American Chemical Society.

Microfluidic perfusion system for automated delivery of temporal gradients to islets of Langerhans.

Science.gov (United States)

Zhang, Xinyu; Roper, Michael G

2009-02-01

A microfluidic perfusion system was developed for automated delivery of stimulant waveforms to cells within the device. The 3-layer glass/polymer device contained two pneumatic pumps, a 12 cm mixing channel, and a 0.2 microL cell chamber. By altering the flow rate ratio of the pumps, a series of output concentrations could be produced while a constant 1.43 +/- 0.07 microL/min flow rate was maintained. The output concentrations could be changed in time producing step gradients and other waveforms, such as sine and triangle waves, at different amplitudes and frequencies. Waveforms were analyzed by comparing the amplitude of output waveforms to the amplitude of theoretical waveforms. Below a frequency of 0.0098 Hz, the output waveforms had less than 20% difference than input waveforms. To reduce backflow of solutions into the pumps, the operational sequence of the valving program was modified, as well as differential etching of the valve seat depths. These modifications reduced backflow to the point that it was not detected. Gradients in glucose levels were applied in this work to stimulate single islets of Langerhans. Glucose gradients between 3 and 20 mM brought clear and intense oscillations of intracellular [Ca(2+)] indicating the system will be useful in future studies of cellular physiology.

The influence of ALN-Al gradient material gradient index on ballistic performance

International Nuclear Information System (INIS)

Wang Youcong; Liu Qiwen; Li Yao; Shen Qiang

2013-01-01

Ballistic performance of the gradient material is superior to laminated material, and gradient materials have different gradient types. Using ls-dyna to simulate the ballistic performance of ALN-AL gradient target plates which contain three gradient index (b = 1, b = 0.5, b = 2). Through Hopkinson bar numerical simulation to the target plate materials, we obtained the reflection stress wave and transmission stress wave state of gradient material to get the best gradient index. The internal stress state of gradient material is simulated by amplification processing of the target plate model. When the gradient index b is equal to 1, the gradient target plate is best of all.

Micromechanical Characterization of Hydrogels Undergoing Swelling and Dissolution at Alkaline pH

Directory of Open Access Journals (Sweden)

Wei Hu

2017-11-01

Full Text Available The swelling of polyelectrolyte hydrogels usually depends on the pH, and if the pH is high enough degradation can occur. A microindentation device was developed to dynamically test these processes in whey protein isolate hydrogels at alkaline pH 7–14. At low alkaline pH the shear modulus decreases during swelling, consistent with rubber elasticity theory, yet when chemical degradation occurs at pH ≥ 11.5 the modulus decreases quickly and extensively. The apparent modulus was constant with the indentation depth when swelling predominates, but gradients were observed when fast chemical degradation occurs at 0.05–0.1 M NaOH. In addition, these profiles were constant with time when dissolution rates are also constant, the first evidence that a swollen layer with steady state mechanical properties is achieved despite extensive dissolution. At >0.5 M NaOH, we provide mechanical evidence showing that most interactions inside the gels are destroyed, gels were very weak and hardly swell, yet they still dissolve very slowly. Microindentation can provide complementary valuable information to study the degradation of hydrogels.

Ménage-à-trois: The ratio of bicarbonate to CO2 and the pH regulate the capacity of neutrophils to form NETs

Directory of Open Access Journals (Sweden)

Christian Maueröder

2016-12-01

Full Text Available In this study we identified and characterized the potential of a high ratio of bicarbonate to CO2 and a moderately alkaline pH to render neutrophils prone to undergo neutrophil extracellular trap (NET formation. Both experimental settings increased the rate of spontaneous NET release and potentiated the NET-inducing capacity of phorbol esters (PMA, ionomycin, monosodium urate and LPS. In contrast, an acidic environment impaired neutrophil extracellular trap formation both spontaneous and induced. Our findings indicate that intracellular alkalinization of neutrophils in response to an alkaline environment leads to an increase of intracellular calcium and neutrophil activation. We further found that the anion channel blocker DIDS strongly reduced NET formation induced by bicarbonate. This finding suggests that the effects observed are due to a molecular program that renders neutrophils susceptible to neutrophil extracellular trap formation. Inflammatory foci are characterized by an acidic environment. Our data indicates that NET formation is favored by the higher pH at the border regions of inflamed areas. Moreover our findings highlight the necessity for strict pH control during assays of neutrophil extracellular trap formation.

Gradient simulation experiments for targeting population heterogeneity in continuous Saccharomyces cerevisiae fermentation

DEFF Research Database (Denmark)

Heins, Anna-Lena; Lencastre Fernandes, Rita; Lundin, L.

Traditionally, a microbial population has been considered homogeneous in optimization studies of fermentation processes. However, research has shown that a typical microbial population in a fermenter is heterogeneous. There are indications that such heterogeneity may be both beneficial (facilitates...... quick adaptation to new conditions) and harmful (reduces yields and productivities) (Bylund et al. (1998); Enfors et al. (2001)). Significant gradients of e.g. dissolved oxygen, substrates, and pH are typically observed in many industrial scale fermentation processes. Consequently, the microbial cells...... reporter strain demonstrated a highly dynamic behaviour with regards to subpopulation distribution during the different growth stages. To simulate which effect glucose gradients, often seen in large scale cultivations, have on population heterogeneity, glucose perturbations during continuous cultivation...

Dependence of Relative Expression of NTR1 and EGFR on Cell Density and Extracellular pH in Human Pancreatic Cancer Cell Lines

International Nuclear Information System (INIS)

Olszewski-Hamilton, Ulrike; Hamilton, Gerhard

2011-01-01

Pancreatic adenocarcinoma is a devastating disease characterized by early dissemination and poor prognosis. These solid tumors express receptors for neuropeptides like neurotensin (NT) or epidermal growth factor (EGF) and exhibit acidic regions when grown beyond a certain size. We previously demonstrated increases in intracellular Ca 2+ levels, intracellular pH and interleukin-8 (IL-8) secretion in BxPC-3 and PANC-1 pancreatic cancer cells in response to a stable NT analog. The present study aimed at investigation of the dependence of the relative expression of NT receptor 1 (NTR1) and EGFR in BxPC-3 and MIA PaCa-2 cells on cell density and extracellular pH (pH e ). MTT assays revealed the NTR1 inhibitor SR 142948-sensitive Lys 8 -ψ-Lys 9 NT (8–13)-induced proliferation in BxPC-3 and PANC-1 cells. Confluent cultures of BxPC3 and HT-29 lines exhibited highest expression of NTR1 and lowest of EGFR and expression of NTR1 was maximal at slightly acidic pH e . IL-8 production was stimulated by Lys 8 -ψ-Lys 9 NT (8–13) and even enhanced at both acidic and alkaline pH e in BxPC-3 and PANC-1 cells. In conclusion, our in vitro study suggests that one contributing factor to the minor responses obtained with EGFR-directed therapy may be downregulation of this receptor in tumor cell aggregates, possibly resulting in acquisition of a more aggressive phenotype via other growth factor receptors like NTR1

Gradient waveform synthesis for magnetic propulsion using MRI gradient coils

International Nuclear Information System (INIS)

Han, B H; Lee, S Y; Park, S

2008-01-01

Navigating an untethered micro device in a living subject is of great interest for both diagnostic and therapeutic applications. Magnetic propulsion of an untethered device carrying a magnetic core in it is one of the promising methods to navigate the device. MRI gradients coils are thought to be suitable for navigating the device since they are capable of magnetic propulsion in any direction while providing magnetic resonance images. For precise navigation of the device, especially in the peripheral region of the gradient coils, the concomitant gradient fields, as well as the linear gradient fields in the main magnetic field direction, should be considered in driving the gradient coils. For simple gradient coil configurations, the Maxwell coil in the z-direction and the Golay coil in the x- and y-directions, we have calculated the magnetic force fields, which are not necessarily the same as the conventional linear gradient fields of MRI. Using the calculated magnetic force fields, we have synthesized gradient waveforms to navigate the device along a desired path

Development of an intracellular glycolytic flux sensor for high throughput applications in E.coli

DEFF Research Database (Denmark)

Lehning, Christina Eva

The aim of this PhD project was to construct, test and apply an intracellular, growth-­‐ independent and direct measureable glycolytic flux biosensor in E. coli. Studying the metabolic flux of bacterial cells is of growing interest as it is of fundamental importance to bacterial physiology as well...... to study the flux-­‐altering effects of gene knockouts in E. coli at the single cell level in a vastly parallelized and high-­‐throughput manner. After growth for several generations in rich and minimal media, 2126 gene knockouts, mainly outside of the core metabolism, could be screened. 3 gene knockouts...

The dependence of fibroblast radiosensitivity on cell pH

International Nuclear Information System (INIS)

Veksler, A.M.; Kublik, L.N.; Degtyareva, O.V.; Ehjdus, L.Kh.

1983-01-01

The problem of the change of radiosensitivity of Chinese hamster fibroblasts, irradiated under aerobic and hypoxic conditions in the course of intracellular pH (pHsub(intr.)) change by means of a phosphate buffer has been studied. It has been found that pHsub(intr.) reduction considerably increases the radiosensitivity, the effect being more pronounced on hypoxic cells which is essential for radiotherapy of tumors. The survival rate of cell irradiated under hypoxia conditions does not depend on season while cell resistance in case of irradiation in open air in spring and autumn is different. The effect discovery in case of pHsub(intr.) reduction upon irradiation shows up the influence of the studied factor on repair processes

Growth and shrinkage of pluronic micelles by uptake and release of flurbiprofen: variation of pH.

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Alexander, Shirin; de Vos, Wiebe M; Castle, Thomas C; Cosgrove, Terence; Prescott, Stuart W

2012-04-24

The micellization of Pluronic triblock copolymers (P103, P123, and L43) in the presence of flurbiprofen at different pH was studied by small-angle neutron scattering (SANS), pulsed-field gradient stimulated-echo nuclear magnetic resonance (PFGSE-NMR), and surface tension measurements. Addition of flurbiprofen to the Pluronic at low pH leads to an increase in the fraction of micellization, aggregation number, and the core radius of the micelles. However, changing the pH to above the pKa of flurbiprofen in an ethanol/water mixture (∼6.5) reduces the fraction of micellization and results in a weaker interaction between the drug and micelles due to the increased drug solubility in aqueous solution.

Self-assembled nanocomplexes of anionic pullulan and polyallylamine for DNA and pH-sensitive intracellular drug delivery

International Nuclear Information System (INIS)

Vora, Lalit; Tyagi, Monica; Patel, Ketan; Gupta, Sanjay; Vavia, Pradeep

2014-01-01

The amalgamation of chemotherapy and gene therapy is promising treatment option for cancer. In this study, novel biocompatible self-assembled nanocomplexes (NCs) between carboxylmethylated pullulan t335 (CMP) with polyallylamine (CMP–PAA NCs) were developed for plasmid DNA (pDNA) and pH-sensitive doxorubicin (DOX) delivery. DOX was conjugated to CMP (DOX–CMP) via hydrazone and confirmed by FTIR and 1 H-NMR. In vitro release studies of pH-sensitive DOX–CMP conjugate showed 23 and 85 % release after 48 h at pH 7.4 (physiological pH) and pH 5 (intracellular/tumoral pH), respectively. The CMP–PAA NCs or DOX–CMP–PAA NCs self-assembled into a nanosized (<250 nm) spherical shape as confirmed by DLS and TEM. The hemolysis and cytotoxicity study indicated that the CMP–PAA NCs did not show cytotoxicity in comparison with plain polyallylamine. Gel retardation assay showed complete binding of pDNA with CMP–PAA NCs at 1:2 weight ratio. CMP–PAA NCs/pDNA showed significantly higher transfection in HEK293 cells compared to PAA/pDNA complexes. Confocal imaging demonstrated successful cellular uptake of DOX–CMP–PAA NCs in HEK293 cells. Thus, NCs hold great potential for targeted pDNA and pH-sensitive intratumoral drug delivery

Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome.

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Kane, Lesley A; Yung, Christina K; Agnetti, Giulio; Neverova, Irina; Van Eyk, Jennifer E

2006-11-01

Separation of basic proteins with 2-DE presents technical challenges involving protein precipitation, load limitations, and streaking. Cardiac mitochondria are enriched in basic proteins and difficult to resolve by 2-DE. We investigated two methods, cup and paper bridge, for sample loading of this subproteome into the basic range (pH 6-11) gels. Paper bridge loading consistently produced improved resolution of both analytical and preparative protein loads. A unique benefit of this technique is that proteins retained in the paper bridge after loading basic gels can be reloaded onto lower pH gradients (pH 4-7), allowing valued samples to be analyzed on multiple pH ranges.

pH sensing by lipids in membranes: The fundamentals of pH-driven migration, polarization and deformations of lipid bilayer assemblies.

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Angelova, Miglena I; Bitbol, Anne-Florence; Seigneuret, Michel; Staneva, Galya; Kodama, Atsuji; Sakuma, Yuka; Kawakatsu, Toshihiro; Imai, Masayuki; Puff, Nicolas

2018-03-06

Most biological molecules contain acido-basic groups that modulate their structure and interactions. A consequence is that pH gradients, local heterogeneities and dynamic variations are used by cells and organisms to drive or regulate specific biological functions including energetic metabolism, vesicular traffic, migration and spatial patterning of tissues in development. While the direct or regulatory role of pH in protein function is well documented, the role of hydrogen and hydroxyl ions in modulating the properties of lipid assemblies such as bilayer membranes is only beginning to be understood. Here, we review approaches using artificial lipid vesicles that have been instrumental in providing an understanding of the influence of pH gradients and local variations on membrane vectorial motional processes: migration, membrane curvature effects promoting global or local deformations, crowding generation by segregative polarization processes. In the case of pH induced local deformations, an extensive theoretical framework is given and an application to a specific biological issue, namely the structure and stability of mitochondrial cristae, is described. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo. Copyright © 2018 Elsevier B.V. All rights reserved.

The Effect of pH and Time on The Stability of Superparamagnetic Maghemite Nanoparticle Suspensions

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Nurdin Irwan

2016-01-01

Full Text Available Maghemite (γ-Fe2O3 nanoparticles have been synthesized using a chemical co-precipitation method. The morphology and particle size is characterized using Transmission Electron Microscopy (TEM, and magnetic characterization using Alternating Gradient Magnetometry (AGM. The stability of the maghemite nanoparticles suspension were studied at different pH and time of storage. Dynamic Light Scattering (DLS and Zeta Potential were conducted to determine the stability of the suspensions. TEM observation showed that the particles size is 9.6 nm and have spherical morphology. The particles showed superparamagnetic behavior with saturation magnetization 25.5 emu/g. The suspensions are stable in the acidic condition at pH 4 and alkaline condition at pH 10. The suspensions remain stable after 4 weeks of storage.

Intracellular trafficking of a pH-responsive drug metal complex.

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Kheirolomoom, Azadeh; Ingham, Elizabeth S; Commisso, Joel; Abushaban, Neveen; Ferrara, Katherine W

2016-12-10

We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1

Combining Step Gradients and Linear Gradients in Density.

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Kumar, Ashok A; Walz, Jenna A; Gonidec, Mathieu; Mace, Charles R; Whitesides, George M

2015-06-16

Combining aqueous multiphase systems (AMPS) and magnetic levitation (MagLev) provides a method to produce hybrid gradients in apparent density. AMPS—solutions of different polymers, salts, or surfactants that spontaneously separate into immiscible but predominantly aqueous phases—offer thermodynamically stable steps in density that can be tuned by the concentration of solutes. MagLev—the levitation of diamagnetic objects in a paramagnetic fluid within a magnetic field gradient—can be arranged to provide a near-linear gradient in effective density where the height of a levitating object above the surface of the magnet corresponds to its density; the strength of the gradient in effective density can be tuned by the choice of paramagnetic salt and its concentrations and by the strength and gradient in the magnetic field. Including paramagnetic salts (e.g., MnSO4 or MnCl2) in AMPS, and placing them in a magnetic field gradient, enables their use as media for MagLev. The potential to create large steps in density with AMPS allows separations of objects across a range of densities. The gradients produced by MagLev provide resolution over a continuous range of densities. By combining these approaches, mixtures of objects with large differences in density can be separated and analyzed simultaneously. Using MagLev to add an effective gradient in density also enables tuning the range of densities captured at an interface of an AMPS by simply changing the position of the container in the magnetic field. Further, by creating AMPS in which phases have different concentrations of paramagnetic ions, the phases can provide different resolutions in density. These results suggest that combining steps in density with gradients in density can enable new classes of separations based on density.

Temperature sensitivity and enzymatic mechanisms of soil organic matter decomposition along an altitudinal gradient on Mount Kilimanjaro

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Blagodatskaya, Еvgenia; Blagodatsky, Sergey; Khomyakov, Nikita; Myachina, Olga; Kuzyakov, Yakov

2016-02-01

Short-term acceleration of soil organic matter decomposition by increasing temperature conflicts with the thermal adaptation observed in long-term studies. Here we used the altitudinal gradient on Mt. Kilimanjaro to demonstrate the mechanisms of thermal adaptation of extra- and intracellular enzymes that hydrolyze cellulose, chitin and phytate and oxidize monomers (14C-glucose) in warm- and cold-climate soils. We revealed that no response of decomposition rate to temperature occurs because of a cancelling effect consisting in an increase in half-saturation constants (Km), which counteracts the increase in maximal reaction rates (Vmax with temperature). We used the parameters of enzyme kinetics to predict thresholds of substrate concentration (Scrit) below which decomposition rates will be insensitive to global warming. Increasing values of Scrit, and hence stronger canceling effects with increasing altitude on Mt. Kilimanjaro, explained the thermal adaptation of polymer decomposition. The reduction of the temperature sensitivity of Vmax along the altitudinal gradient contributed to thermal adaptation of both polymer and monomer degradation. Extrapolating the altitudinal gradient to the large-scale latitudinal gradient, these results show that the soils of cold climates with stronger and more frequent temperature variation are less sensitive to global warming than soils adapted to high temperatures.

Gastrointestinal pH profile in subjects with irritable bowel syndrome.

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Lalezari, David

2012-01-01

To investigate the small bowel pH profile and small intestine transit time (SITT) in healthy controls and patients with irritable bowel syndrome (IBS). Nine IBS patients (3 males, mean age 35 yr) and 10 healthy subjects (6 males, mean age 33 yr) were studied. Intestinal pH profile and SITT were assessed by a wireless motility pH and pressure capsule (Smart Pill). Mean pH values were measured in the small intestine (SI) and compared both within and between groups. Data presented as mean or median, ANOVA, P <0.05 for significance. We found the pH for the first (Q1), second (Q2), third (Q3), and fourth quartile (Q4) of the SI in healthy versus IBS patients was 5.608 ± 0.491 vs. 5.667 ± 0.297, 6.200 ± 0.328 vs. 6.168 ± 0.288, 6.679 ± 0.316 vs. 6.741 ± 0.322, and 6.884 ± 0.200 vs. 6.899 ± 0.303, respectively. We found no significant group difference in pH per quartile (P=0.7979). The proximal SI was significantly more acidic, compared to distal segments, in both healthy subjects and IBS patients (P<0.0001). We found no significant difference in the measured SITT between IBS and control groups with a mean SITT of 218.56 ± 59.60 min and 199.20 ± 82.31 min, respectively (P=0.55). This study shows the presence of a gradient of pH along the SI, in both IBS and healthy subjects, the distal being less acidic. These finding may be of importance in small bowel homeostasis.

Transcription factors Asg1p and Hal9p regulate pH homeostasis in Candida glabrata

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Jing eWu

2015-08-01

Full Text Available Candida glabrata is an important microorganism used in commercial fermentation to produce pyruvate, but very little is known about its mechanisms for surviving acid stress in culture. In this study, it was shown that transcription factors Asg1p and Hal9p play essential roles in C. glabrata in the tolerance of acid stress, as the deletion of CgASG1 or CgHAL9 resulted in the inability to survive in an acidic environment. Cgasg1 and Cghal9 mutant strains are unable to maintain pH homeostasis, as evidenced by a decrease in intracellular pH and an increase in reactive oxygen species production, which results in metabolic disorders. The results showed that intracellular acidification was partly due to the diminished activity of the plasma membrane proton pump, CgPma1p. In addition, transcriptome sequencing revealed that Cgasg1 and Cghal9 mutant strains displayed a variety of changes in gene expression under acidic conditions, including genes in the MAPK signaling pathway, plasma membrane or cell wall organization, trehalose accumulation, and the RIM101 signaling pathway. Lastly, quantitative reverse-transcribed PCR and cellular localization showed that CgAsg1p and CgHal9p played independent roles in response to acid stress.

Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells

International Nuclear Information System (INIS)

Sun, Y H; Liu, Y S; Vernier, P T; Liang, C H; Chong, S Y; Marcu, L; Gundersen, M A

2006-01-01

Photophysical properties of semiconductor nanocrystal quantum dots (QDs) are primary determinants of their efficacy as fluorescence probes in biological systems. Our minimally passivated core/shell/shell QDs are smaller than the QDs with thick polymer coats that are often used for cellular probes, permitting less restricted access to intracellular compartments and at the same time a greater sensitivity to environmental conditions. We report here a reversible photoinduced fluorescence enhancement (photoactivation) of endocytosed mercaptoacetic-acid-capped CdSe quantum dots (MAA QDs) and the pH dependence of MAA QD photoluminescence in SKOV-3 human ovarian cancer cells. The fluorescence emission of MAA QDs taken up directly by SKOV-3 cells without the need for extra capping ligands or permeabilization steps remains bright and stable for at least 14 days. These intracellular fluorescent nanocrystals do not colocalize with low-pH lysosomes, and the emission of the MAA QDs in fixed cell preparations is quenched by acidic buffer, suggesting that a low-pH environment in cellular vesicles quenches QD fluorescence. Photoactivation of intracellular MAA QD luminescence is dependent on the excitation energy and is related to the metabolic activity of the cells. These active interactions between cells and nanocrystals demonstrate the potential of MAA QDs as intracellular environmental sensors

Intracellular transport driven by cytoskeletal motors: General mechanisms and defects

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Appert-Rolland, C.; Ebbinghaus, M.; Santen, L.

2015-09-01

Cells are the elementary units of living organisms, which are able to carry out many vital functions. These functions rely on active processes on a microscopic scale. Therefore, they are strongly out-of-equilibrium systems, which are driven by continuous energy supply. The tasks that have to be performed in order to maintain the cell alive require transportation of various ingredients, some being small, others being large. Intracellular transport processes are able to induce concentration gradients and to carry objects to specific targets. These processes cannot be carried out only by diffusion, as cells may be crowded, and quite elongated on molecular scales. Therefore active transport has to be organized. The cytoskeleton, which is composed of three types of filaments (microtubules, actin and intermediate filaments), determines the shape of the cell, and plays a role in cell motion. It also serves as a road network for a special kind of vehicles, namely the cytoskeletal motors. These molecules can attach to a cytoskeletal filament, perform directed motion, possibly carrying along some cargo, and then detach. It is a central issue to understand how intracellular transport driven by molecular motors is regulated. The interest for this type of question was enhanced when it was discovered that intracellular transport breakdown is one of the signatures of some neuronal diseases like the Alzheimer. We give a survey of the current knowledge on microtubule based intracellular transport. Our review includes on the one hand an overview of biological facts, obtained from experiments, and on the other hand a presentation of some modeling attempts based on cellular automata. We present some background knowledge on the original and variants of the TASEP (Totally Asymmetric Simple Exclusion Process), before turning to more application oriented models. After addressing microtubule based transport in general, with a focus on in vitro experiments, and on cooperative effects in the

Lysosomal and endosomal heterogeneity in the liver: A comparison of the intracellular pathways of endocytosis in rat liver cells

International Nuclear Information System (INIS)

Kindberg, G.M.; Tolleshaug, H.; Gjoen, T.; Berg, T.

1991-01-01

Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients

Intermittent Hypoxia Inhibits Na+-H+ Exchange-Mediated Acid Extrusion Via Intracellular Na+ Accumulation in Cardiomyocytes

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Huai-Ren Chang

2018-04-01

Full Text Available Background/Aims: Intermittent hypoxia (IH has been shown to exert preconditioning-like cardioprotective effects. It also has been reported that IH preserves intracellular pH (pHi during ischemia and protects cardiomyocytes against ischemic reperfusion injury. However, the exact mechanism is still unclear. Methods: In this study, we used proton indicator BCECF-AM to analyze the rate of pHi recovery from acidosis in the IH model of rat neonatal cardiomyocytes. Neonatal cardiomyocytes were first treated with repetitive hypoxia-normoxia cycles for 1-4 days. Cells were then acid loaded with NH4Cl, and the rate of pHi recovery from acidosis was measured. Results: We found that the pHi recovery rate from acidosis was much slower in the IH group than in the room air (RA group. When we treated cardiomyocytes with Na+-H+ exchange (NHE inhibitors (Amiloride and HOE642 or Na+-free Tyrode solution during the recovery, there was no difference between RA and IH groups. We also found intracellular Na+ concentration ([Na+]i significantly increased after IH exposure for 4 days. However, the phenomenon could be abolished by pretreatment with ROS inhibitors (SOD and phenanathroline, intracellular calcium chelator or Na+-Ca2+ exchange (NCX inhibitor. Furthermore, the pHi recovery rate from acidosis became faster in the IH group than in the RA group when inhibition of NCX activity. Conclusions: These results suggest that IH would induce the elevation of ROS production. ROS then activates Ca2+-efflux mode of NCX and results in intracellular Na+ accumulation. The rise of [Na+]i further inhibits the activity of NHE-mediated acid extrusion and retards the rate of pHi recovery from acidosis during IH.

pH control and rapid mixing in spinning NMR samples

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Yesinowski, James P.; Sunberg, Richard J.; Benedict, James J.

An apparatus is described which permits the acquisition of NMR spectra from spinning 20-mm sample tubes while: (1) constantly monitoring the pH; (2) adding reagents to maintain constant pH (pH-statting); (3) efficiently mixing the added reagent. The apparatus was built to study the spontaneous precipitation of calcium phosphates from supersaturated solutions using 31P NMR. Other applications include the rapid determination of NMR titration curves, and the minimization of temperature gradients in large sample tubes. The apparatus was used to measure the 31P chemical shift titration of dilute phosphoric acid, which yielded accurate shifts for the three species of protonated orthophosphate ion. The bulk magnetic susceptibility of 85% H 3PO 4 relative to a dilute aqueous sample was also measured, and is shown to contribute significantly to chemical shift measurements.

Water balance creates a threshold in soil pH at the global scale

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Slessarev, E. W.; Lin, Y.; Bingham, N. L.; Johnson, J. E.; Dai, Y.; Schimel, J. P.; Chadwick, O. A.

2016-12-01

Soil pH regulates the capacity of soils to store and supply nutrients, and thus contributes substantially to controlling productivity in terrestrial ecosystems. However, soil pH is not an independent regulator of soil fertility—rather, it is ultimately controlled by environmental forcing. In particular, small changes in water balance cause a steep transition from alkaline to acid soils across natural climate gradients. Although the processes governing this threshold in soil pH are well understood, the threshold has not been quantified at the global scale, where the influence of climate may be confounded by the effects of topography and mineralogy. Here we evaluate the global relationship between water balance and soil pH by extracting a spatially random sample (n = 20,000) from an extensive compilation of 60,291 soil pH measurements. We show that there is an abrupt transition from alkaline to acid soil pH that occurs at the point where mean annual precipitation begins to exceed mean annual potential evapotranspiration. We evaluate deviations from this global pattern, showing that they may result from seasonality, climate history, erosion and mineralogy. These results demonstrate that climate creates a nonlinear pattern in soil solution chemistry at the global scale; they also reveal conditions under which soils maintain pH out of equilibrium with modern climate.

Momentum-weighted conjugate gradient descent algorithm for gradient coil optimization.

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Lu, Hanbing; Jesmanowicz, Andrzej; Li, Shi-Jiang; Hyde, James S

2004-01-01

MRI gradient coil design is a type of nonlinear constrained optimization. A practical problem in transverse gradient coil design using the conjugate gradient descent (CGD) method is that wire elements move at different rates along orthogonal directions (r, phi, z), and tend to cross, breaking the constraints. A momentum-weighted conjugate gradient descent (MW-CGD) method is presented to overcome this problem. This method takes advantage of the efficiency of the CGD method combined with momentum weighting, which is also an intrinsic property of the Levenberg-Marquardt algorithm, to adjust step sizes along the three orthogonal directions. A water-cooled, 12.8 cm inner diameter, three axis torque-balanced gradient coil for rat imaging was developed based on this method, with an efficiency of 2.13, 2.08, and 4.12 mT.m(-1).A(-1) along X, Y, and Z, respectively. Experimental data demonstrate that this method can improve efficiency by 40% and field uniformity by 27%. This method has also been applied to the design of a gradient coil for the human brain, employing remote current return paths. The benefits of this design include improved gradient field uniformity and efficiency, with a shorter length than gradient coil designs using coaxial return paths. Copyright 2003 Wiley-Liss, Inc.

On the intracellular release mechanism of hydrophobic cargo and its relation to the biodegradation behavior of mesoporous silica nanocarriers.

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von Haartman, Eva; Lindberg, Desiré; Prabhakar, Neeraj; Rosenholm, Jessica M

2016-12-01

The intracellular release mechanism of hydrophobic molecules from surface-functionalized mesoporous silica nanoparticles was studied in relation to the biodegradation behavior of the nanocarrier, with the purpose of determining the dominant release mechanism for the studied drug delivery system. To be able to follow the real-time intracellular release, a hydrophobic fluorescent dye was used as model drug molecule. The in vitro release of the dye was investigated under varying conditions in terms of pH, polarity, protein and lipid content, presence of hydrophobic structures and ultimately, in live cancer cells. Results of investigating the drug delivery system show that the degradation and drug release mechanisms display a clear interdependency in simple aqueous solvents. In pure aqueous media, the cargo release was primarily dependent on the degradation of the nanocarrier, while in complex media, mimicking intracellular conditions, the physicochemical properties of the cargo molecule itself and its interaction with the carrier and/or surrounding media were found to be the main release-governing factors. Since the material degradation was retarded upon loading with hydrophobic guest molecules, the cargo could be efficiently delivered into live cancer cells and released intracellularly without pronounced premature release under extracellular conditions. From a rational design point of view, pinpointing the interdependency between these two processes can be of paramount importance considering future applications and fundamental understanding of the drug delivery system. Copyright © 2016 Elsevier B.V. All rights reserved.

Regularities of development of unspecific reaction of cells, and modification of chemical protection

International Nuclear Information System (INIS)

Veksler, A.M.; Korystov, Yu.N.; Kublik, L.N.; Ehjdus, L.Kh.

1979-01-01

Regularities of development of a unspecific reaction of cells under the effect of different substances belonging to weak electrolytes have been studied. It was demonstrated that the rate of the unspecific reaction development under the effect of cysteamine and caffeine-benzoate depends on the agent concentration, temperature and pH of a medium. It was established that the response of a cell is determined by the overall intracellular concentration of the agent rather than by its specific character. The total concentration of the substance inside the cell depends on its physico-chemical characteristics and, with a pH gradient between cell and medium, can markedly vary from that in the medium. With similar intracellular content, both substances proved to be virtually equally effective. This suggests that it is possible to assess the effectiveness of some other biologically active substances many of which are weak electrolytes