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Sample records for acidic intracellular ph

  1. Intracellular pH of acid-tolerant ruminal bacteria.

    PubMed Central

    Russell, J B

    1991-01-01

    Acid-tolerant ruminal bacteria (Bacteroides ruminicola B1(4), Selenomonas ruminantium HD4, Streptococcus bovis JB1, Megasphaera elsdenii B159, and strain F) allowed their intracellular pH to decline as a function of extracellular pH and did not generate a large pH gradient across the cell membrane until the extracellular pH was low (less than 5.2). This decline in intracellular pH prevented an accumulation of volatile fatty acid anions inside the cells. PMID:1781695

  2. Intracellular pH Recovery Rates in Bivalve Hemocytes Following Exposure to Acidic Environmental Conditions

    NASA Astrophysics Data System (ADS)

    Croxton, A.; Wikfors, G. H.

    2012-12-01

    Predictions of ocean acidification effects upon carbonate shell-forming species have caused great concern for the future of shellfisheries. Nevertheless, bivalve species inhabiting an estuarine environment have evolved in these environments with fluctuating pH levels. Previous experimental studies conducted in our laboratory have demonstrated the ability of oyster hemocytes to maintain intracellular homeostasis under acidic external conditions. However, little information is known of this homeostatic mechanism in other molluscan shellfish species present in these same habitats. In the current study we propose to determine if other bivalve species of aquaculture interest also possess this intracellular regulation by applying an in vitro hemocyte pH-recovery assay, previously developed for oysters, on the northern quahog, Mercenaria mercenaria, the blue mussel, Mytilus edulis, and the softshell clam, Mya arenaria. Preliminary results from the determination of initial intracellular pH levels, the initial step in the rate recovery assay, indicated a pH range between 7.0-7.4. This range was comparable to initial values measured in oysters, and consistent with data reported in the current literature. The second step of the hemocyte pH-recovery assay involves exposing oyster hemocytes to acidic external conditions and measuring the ability of the hemocyte intracellular pH to maintain homeostasis (i.e. recovery rate). Results from the recovery rate process will be presented.

  3. Intracellular pH Recovery Rates of Hemocytes from Estuarine and Open Ocean Bivalve Species Following In vitro Acid Challenge

    NASA Astrophysics Data System (ADS)

    Croxton, A.; Wikfors, G.

    2013-12-01

    Decreasing pH in estuarine systems is a growing concern for researchers studying mollusk species. Debates continue on whether estuarine bivalve species are more or less vulnerable to ocean acidification than marine species because estuaries can present multiple environmental stressors. The aim of this study is to understand the homeostatic mechanisms of bivalve hemocytes following exposure to extracellular acid treatment. Previous measurements using fluorescent SNARF probes and flow-cytometry have determined the intracellular pH of hemocytes from several bivalve species (eastern oyster, bay scallop, northern quahog, soft-shell clam, and blue mussel) to range between 7.0-7.4. In the present study of four bivalve species, recovery rate profiles were determined for intracellular hemocyte pH following addition of acid to hemolymph in vitro. These profiles indicate that soft-shell clams and bay scallops maintained homeostasis with very little change in intracellular pH. In contrast, an initial drop in intracellular pH in northern quahogs was followed by a steady recovery of intracellular pH. Contrasting results between species appear to be unrelated to mineral shell composition (aragonite vs. calcite) or habitat location (infaunal vs. epifaunal). The next phase of this study will be to determine if offshore species (surfclams and sea scallops) will have similar responses. Results from these studies will provide a better understanding of the physiological responses of estuarine and marine species exposed to acidified environments.

  4. Acidic intracellular pH shift during Caenorhabditis elegans larval development

    SciTech Connect

    Wadsworth, W.G.; Riddle, D.L. )

    1988-11-01

    During recovery from the developmentally arrested, nonfeeding dauer stage of the nemotode Caenorhabditis elegans, metabolic activation is accompanied by a decrease in intracellular pH (pH{sub i}). Phosphorus-31 nuclear magnetic resonance ({sup 31}P NMR) analyses of perchloric acid extracts show that inorganic phosphate predominates in dauer larvae, whereas ATP and other high-energy metabolites are abundant within 6 hr after dauer larvae have been placed in food to initiate development. Although metabolic activation has been associated with an alkaline pH{sub i} shift in other organisms, in vivo {sup 31}P NMR analysis of recovering dauer larvae shows a pH{sub i} decrease from {approx} 7.3 to {approx} 6.3 within 3 hr after the animals encounter food. This shift occurs before feeding begins, and it coincides with, or soon follows, the developmental commitment to recover from the dauer stage, suggesting that control of pH{sub i} may be important in the regulation of larval development in nematodes.

  5. Resistance of Streptococcus bovis to acetic acid at low pH: Relationship between intracellular pH and anion accumulation

    SciTech Connect

    Russell, J.B. )

    1991-01-01

    Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grown at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). Y{sub ATP} (grams of cells per mole at ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up ({sup 14}C)acetate and ({sup 14}C)benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation.

  6. Resistance of Streptococcus bovis to acetic acid at low pH: relationship between intracellular pH and anion accumulation.

    PubMed Central

    Russell, J B

    1991-01-01

    Streptococcus bovis JB1, an acid-tolerant ruminal bacterium, was able to grow at pHs from 6.7 to 4.5, and 100 mM acetate had little effect on growth rate or proton motive force across the cell membrane. When S. bovis was grown in glucose-limited chemostats at pH 5.2, the addition of sodium acetate (as much as 100 mM) had little effect on the production of bacterial protein. At higher concentrations of sodium acetate (100 to 360 mM), production of bacterial protein declined, but this decrease could largely be explained by a shift in fermentation products (acetate, formate, and ethanol production to lactate production) and a decline in ATP production (3 ATP per glucose versus 2 ATP per glucose). YATP (grams of cells per mole of ATP) was not decreased significantly even by high concentrations of acetate. Cultures supplemented with 100 mM sodium acetate took up [14C]acetate and [14C]benzoate in accordance with the Henderson-Hasselbalch equation and gave similar estimates of intracellular pH. As the extracellular pH declined, S. bovis allowed its intracellular pH to decrease and maintained a relatively constant pH gradient across the cell membrane (0.9 unit). The decrease in intracellular pH prevented S. bovis from accumulating large amounts of acetate anion. On the basis of these results it did not appear that acetate was acting as an uncoupler. The sensitivity of other bacteria to volatile fatty acids at low pH is explained most easily by a high transmembrane pH gradient and anion accumulation. PMID:2036013

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

    SciTech Connect

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

    2010-06-01

    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{sub 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{sub 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{sub 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{sup +}/H{sup +}-antiporter, corroborating an important role of the pH{sub i} in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH{sub i} may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

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

    PubMed

    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. PMID:27091863

  9. Listeria monocytogenes varies among strains to maintain intracellular pH homeostasis under stresses by different acids as analyzed by a high-throughput microplate-based fluorometry

    PubMed Central

    Cheng, Changyong; Yang, Yongchun; Dong, Zhimei; Wang, Xiaowen; Fang, Chun; Yang, Menghua; Sun, Jing; Xiao, Liya; Fang, Weihuan; Song, Houhui

    2015-01-01

    Listeria monocytogenes, a food-borne pathogen, has the capacity to maintain intracellular pH (pHi) homeostasis in acidic environments, but the underlying mechanisms remain elusive. Here, we report a simple microplate-based fluorescent method to determine pHi of listerial cells that were prelabeled with the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester and subjected to acid stress. We found that L. monocytogenes responds differently among strains toward organic and inorganic acids to maintain pHi homeostasis. The capacity of L. monocytogenes to maintain pHi at extracellular pH 4.5 (pHex) was compromised in the presence of acetic acid and lactic acid, but not by hydrochloric acid and citric acid. Organic acids exhibited more inhibitory effects than hydrochloric acid at certain pH conditions. Furthermore, the virulent stains L. monocytogenes EGDe, 850658 and 10403S was more resistant to acidic stress than the avirulent M7 which showed a defect in maintaining pHi homeostasis. Deletion of sigB, a stress-responsive alternative sigma factor from 10403S, markedly altered intracellular pHi homeostasis, and showed a significant growth and survival defect under acidic conditions. Thus, this work provides new insights into bacterial survival mechanism to acidic stresses. PMID:25667585

  10. Histone Acetylation Regulates Intracellular pH

    PubMed Central

    McBrian, Matthew A.; Behbahan, Iman Saramipoor; Ferrari, Roberto; Su, Trent; Huang, Ta-Wei; Li, Kunwu; Hong, Candice S.; Christofk, Heather R.; Vogelauer, Maria; Seligson, David B.; Kurdistani, Siavash K.

    2014-01-01

    SUMMARY Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases as pHi rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pHi with important implications for mechanism of action and therapeutic use of HDAC inhibitors. PMID:23201122

  11. Intracellular pH measurements using perfluorocarbon nanoemulsions

    PubMed Central

    Patrick, Michael J.; Janjic, Jelena M.; Teng, Haibing; O’Hear, Meredith R.; Brown, Cortlyn W.; Stokum, Jesse A.; Schmidt, Brigitte F.; Ahrens, Eric T.; Waggoner, Alan S.

    2014-01-01

    We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon co-incubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions where characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry, and displayed a steady decrease in pH to a level of 5.5 over 3 hours, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using 19F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics and nanoemulsion stability and cell viability over time. PMID:24266634

  12. Intracellular pH measurements using perfluorocarbon nanoemulsions.

    PubMed

    Patrick, Michael J; Janjic, Jelena M; Teng, Haibing; O'Hear, Meredith R; Brown, Cortlyn W; Stokum, Jesse A; Schmidt, Brigitte F; Ahrens, Eric T; Waggoner, Alan S

    2013-12-11

    We report the synthesis and formulation of unique perfluorocarbon (PFC) nanoemulsions enabling intracellular pH measurements in living cells via fluorescent microscopy and flow cytometry. These nanoemulsions are formulated to readily enter cells upon coincubation and contain two cyanine-based fluorescent reporters covalently bound to the PFC molecules, specifically Cy3-PFC and CypHer5-PFC conjugates. The spectral and pH-sensing properties of the nanoemulsions were characterized in vitro and showed the unaltered spectral behavior of dyes after formulation. In rat 9L glioma cells loaded with nanoemulsion, the local pH of nanoemulsions was longitudinally quantified using optical microscopy and flow cytometry and displayed a steady decrease in pH to a level of 5.5 over 3 h, indicating rapid uptake of nanoemulsion to acidic compartments. Overall, these reagents enable real-time optical detection of intracellular pH in living cells in response to pharmacological manipulations. Moreover, recent approaches for in vivo cell tracking using magnetic resonance imaging (MRI) employ intracellular PFC nanoemulsion probes to track cells using (19)F MRI. However, the intracellular fate of these imaging probes is poorly understood. The pH-sensing nanoemulsions allow the study of the fate of the PFC tracer inside the labeled cell, which is important for understanding the PFC cell loading dynamics, nanoemulsion stability and cell viability over time. PMID:24266634

  13. Intracellular pH in Sperm Physiology

    PubMed Central

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

    2014-01-01

    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 Ca2+ 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. PMID:24887564

  14. Intracellular pH of symbiotic dinoflagellates

    NASA Astrophysics Data System (ADS)

    Gibbin, E. M.; Davy, S. K.

    2013-09-01

    Intracellular pH (pHi) is likely to play a key role in maintaining the functional success of cnidarian-dinoflagellate symbiosis, yet until now the pHi of the symbiotic dinoflagellates (genus Symbiodinium) has never been quantified. Flow cytometry was used in conjunction with the ratiometric fluorescent dye BCECF to monitor changes in pHi over a daily light/dark cycle. The pHi of Symbiodinium type B1 freshly isolated from the model sea anemone Aiptasia pulchella was 7.25 ± 0.01 (mean ± SE) in the light and 7.10 ± 0.02 in the dark. A comparable effect of irradiance was seen across a variety of cultured Symbiodinium genotypes (types A1, B1, E1, E2, F1, and F5) which varied between pHi 7.21-7.39 in the light and 7.06-7.14 in the dark. Of note, there was a significant genotypic difference in pHi, irrespective of irradiance.

  15. Intracellular pH responses in the industrially important fungus Trichoderma reesei.

    PubMed

    Valkonen, Mari; Penttilä, Merja; Benčina, Mojca

    2014-09-01

    Preserving an optimal intracellular pH is critical for cell fitness and productivity. The pH homeostasis of the industrially important filamentous fungus Trichoderma reesei (Hypocrea jecorina) is largely unexplored. We analyzed the impact of growth conditions on regulation of intracellular pH of the strain Rut-C30 and the strain M106 derived from the Rut-C30 that accumulates L-galactonic acid-from provided galacturonic acid-as a consequence of L-galactonate dehydratase deletion. For live-cell measurements of intracellular pH, we used the genetically encoded ratiometric pH-sensitive fluorescent protein RaVC. Glucose and lactose, used as carbon sources, had specific effects on intracellular pH of T. reesei. The growth in lactose-containing medium extensively acidified cytosol, while intracellular pH of hyphae cultured in a medium with glucose remained at a higher level. The strain M106 maintained higher intracellular pH in the presence of D-galacturonic acid than its parental strain Rut-C30. Acidic external pH caused significant acidification of cytosol. Altogether, the pH homeostasis of T. reesei Rut-C30 strain is sensitive to extracellular pH and the degree of acidification depends on carbon source. PMID:25046860

  16. Modeling the effects of sodium chloride, acetic acid and intracellular pH on the survival of Escherichia coli O157:H7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primar...

  17. Preferential intracellular pH regulation: hypotheses and perspectives.

    PubMed

    Shartau, Ryan B; Baker, Daniel W; Crossley, Dane A; Brauner, Colin J

    2016-08-01

    The regulation of vertebrate acid-base balance during acute episodes of elevated internal PCO2  is typically characterized by extracellular pH (pHe) regulation. Changes in pHe are associated with qualitatively similar changes in intracellular tissue pH (pHi) as the two are typically coupled, referred to as 'coupled pH regulation'. However, not all vertebrates rely on coupled pH regulation; instead, some preferentially regulate pHi against severe and maintained reductions in pHe Preferential pHi regulation has been identified in several adult fish species and an aquatic amphibian, but never in adult amniotes. Recently, common snapping turtles were observed to preferentially regulate pHi during development; the pattern of acid-base regulation in these species shifts from preferential pHi regulation in embryos to coupled pH regulation in adults. In this Commentary, we discuss the hypothesis that preferential pHi regulation may be a general strategy employed by vertebrate embryos in order to maintain acid-base homeostasis during severe acute acid-base disturbances. In adult vertebrates, the retention or loss of preferential pHi regulation may depend on selection pressures associated with the environment inhabited and/or the severity of acid-base regulatory challenges to which they are exposed. We also consider the idea that the retention of preferential pHi regulation into adulthood may have been a key event in vertebrate evolution, with implications for the invasion of freshwater habitats, the evolution of air breathing and the transition of vertebrates from water to land. PMID:27489212

  18. Mycothiol protects Corynebacterium glutamicum against acid stress via maintaining intracellular pH homeostasis, scavenging ROS, and S-mycothiolating MetE.

    PubMed

    Liu, Yingbao; Yang, Xiaobing; Yin, Yajie; Lin, Jinshui; Chen, Can; Pan, Junfeng; Si, Meiru; Shen, Xihui

    2016-07-14

    Mycothiol (MSH) plays a major role in protecting cells against oxidative stress and detoxification from a broad range of exogenous toxic agents. In the present study, we reveal that intracellular MSH contributes significantly to the adaptation to acidic conditions in the model organism Corynebacterium glutamicum. We present evidence that MSH confers C. glutamicum with the ability to adapt to acidic conditions by maintaining pHi homeostasis, scavenging reactive oxygen species (ROS), and protecting methionine synthesis by the S-mycothiolation modification of methionine synthase (MetE). The role of MSH in acid adaptation was further confirmed by improving the acid tolerance of C. glutamicum by overexpressing the key MSH synthesis gene mshA. Hence, our work provides insights into a previously unknown, but important, aspect of the C. glutamicum cellular response to acid stress. The results reported here may help to understand acid tolerance mechanisms in acid sensitive bacteria and may open a new avenue for improving acid resistance in industry strains for the production of bio-based chemicals from renewable biomass. PMID:27250661

  19. Computer model of unstirred layer and intracellular pH changes. Determinants of unstirred layer pH.

    PubMed

    Marrannes, Roger

    2013-06-01

    Transmembrane acid-base fluxes affect the intracellular pH and unstirred layer pH around a superfused biological preparation. In this paper the factors influencing the unstirred layer pH and its gradient are studied. An analytical expression of the unstirred layer pH gradient in steady state is derived as a function of simultaneous transmembrane fluxes of (weak) acids and bases with the dehydration reaction of carbonic acid in equilibrium. Also a multicompartment computer model is described consisting of the extracellular bulk compartment, different unstirred layer compartments and the intracellular compartment. With this model also transient changes and the influence of carbonic anhydrase (CA) can be studied. The analytical expression and simulations with the multicompartment model demonstrate that in steady state the unstirred layer pH and its gradient are influenced by the size and type of transmembrane flux of acids and bases, their dissociation constant and diffusion coefficient, the concentration, diffusion coefficient and type of mobile buffers and the activity and location of CA. Similar principles contribute to the amplitude of the unstirred layer pH transients. According to these models an immobile buffer does not influence the steady-state pH, but reduces the amplitude of pH transients especially when these are fast. The unstirred layer pH provides useful information about transmembrane acid-base fluxes. This paper gives more insight how the unstirred layer pH and its transients can be interpreted. Methodological issues are discussed. PMID:23860924

  20. Altered intracellular pH regulation in cells with high levels of P-glycoprotein expression.

    PubMed

    Young, Gregory; Reuss, Luis; Altenberg, Guillermo A

    2011-01-01

    P-glycoprotein is an ATP-binding-cassette transporter that pumps many structurally unrelated drugs out of cells through an ATP-dependent mechanism. As a result, multidrug-resistant cells that overexpress P-glycoprotein have reduced intracellular steady-state levels of a variety of chemotherapeutic agents. In addition, increased cytosolic pH has been a frequent finding in multidrug-resistant cells that express P-glycoprotein, and it has been proposed that this consequence of P-glycoprotein expression may contribute to the lower intracellular levels of chemotherapeutic agents. In these studies, we measured intracellular pH and the rate of acid extrusion in response to an acid load in two cells with very different levels of P-glycoprotein expression: V79 parental cells and LZ-8 multidrug resistant cells. Compared to the wild-type V79 cells, LZ-8 cells have a lower intracellular pH and a slower recovery of intracellular pH after an acid load. The data also show that LZ-8 cells have reduced ability to extrude acid, probably due to a decrease in Na(+)/H(+) exchanger activity. The alterations in intracellular pH and acid extrusion in LZ-8 cells are reversed by 24-h exposure to the multidrug-resistance modulator verapamil. The lower intracellular pH in LZ-8 indicates that intracellular alkalinization is not necessary for multidrug resistance. The reversal by verapamil of the decreased acid-extrusion suggests that P-glycoprotein can affect other membrane transport mechanism. PMID:22003434

  1. A polymer-Triton X-100 conjugate capable of PH-dependent red blood cell lysis: a model system illustrating the possibility of drug delivery within acidic intracellular compartments.

    PubMed

    Duncan, R; Ferruti, P; Sgouras, D; Tuboku-Metzger, A; Ranucci, E; Bignotti, F

    1994-01-01

    Poly(amidoamines) are soluble polymers containing tertiary amino and amido groups regularly arranged along the macromolecular chain, and their net average charge alters considerably as pH changes from neutral to acidic leading to a change in conformation. This property provides the possibility to design polymer-drug conjugates that are, following intravenous administration, relatively compacted and thus protect a drug payload in the circulation, but following pinocytic internalisation into acidic intracellular compartments unfold permitting pH-triggered intracellular drug delivery. To study the feasibility of this approach, a covalent conjugate of a poly(amidoamine) (MBI) was prepared to contain the membrane lytic non-ionic detergent Triton X-100 (as a model), and its ability to lyse red blood cells in vitro was used as an indicator of conjugate conformation at at different pHs. Although Triton X-100 was highly lytic at pH 5.5, 7.4 and 8.0, and the parent polymer MBI was not lytic under any conditions, the conjugate only showed concentration-dependent red blood cell lysis at pH 5.5. Moreover, incubation of human leukaemic cells (CCRF) with these substrates showed conjugate to be more toxic than MBI (IC50 values of 100 micrograms/ml and 650 micrograms/ml respectively) and less toxic than Triton X-100 (IC50 of 1 microgram/ml). PMID:7858959

  2. Regulation of lung surfactant secretion by intracellular pH.

    PubMed

    Chander, A

    1989-12-01

    We investigated secretion of lung surfactant phosphatidylcholine (PC) using isolated perfused rat lung preparation after labeling the lung lipids in vitro with [methyl-3H]choline. The perfusion medium was Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM glucose and 3% fatty acid-poor bovine serum albumin. After ventilation of lungs with air containing 5% CO2 (control) for 1 h, 0.91% +/- 0.04 (mean +/- SE, n = 6) of total lung lipid radioactivity (greater than 95% in PC) was recovered in the cell-free lavage fluid. The secretion of PC was increased with terbutaline (50 microM), 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 100 microM), phorbol L2-myristate 13-acetate (30 ng/ml), and ATP (1 mM), in each case by approximately 150%. Secretion of PC was also increased by 160% if the lungs were ventilated with air containing 0% CO2. The low CO2-mediated PC secretion was time and concentration dependent. The dose-response curve for 0-10% CO2 was S-shaped. The low CO2-induced increase in PC secretion could be largely reversed with diffusible weak acids (25 mM, acetate or butyrate) in the perfusion medium. An increase (70%) in secretion was also induced with 10 mM NH4Cl, suggesting a role for intracellular alkalosis. These observations suggest that intracellular alkalosis stimulates lung surfactant secretion. Alkalosis-stimulated secretion of PC was additive with that with terbutaline (5 X 10(-7) to 5 X 10(-4) M) or 10(-4) M 8-BrcAMP, suggesting that alkalosis effect was not mediated through the beta-adrenergic pathway of surfactant secretion.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2514603

  3. Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing.

    PubMed

    Shangguan, Jingfang; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Liu, Jinquan; Tang, Jinlu; Yang, Xue; Huang, Jin

    2016-08-01

    Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels. PMID:27334762

  4. Intracellular pH Modulates Autophagy and Mitophagy.

    PubMed

    Berezhnov, Alexey V; Soutar, Marc P M; Fedotova, Evgeniya I; Frolova, Maria S; Plun-Favreau, Helene; Zinchenko, Valery P; Abramov, Andrey Y

    2016-04-15

    The specific autophagic elimination of mitochondria (mitophagy) plays the role of quality control for this organelle. Deregulation of mitophagy leads to an increased number of damaged mitochondria and triggers cell death. The deterioration of mitophagy has been hypothesized to underlie the pathogenesis of several neurodegenerative diseases, most notably Parkinson disease. Although some of the biochemical and molecular mechanisms of mitochondrial quality control are described in detail, physiological or pathological triggers of mitophagy are still not fully characterized. Here we show that the induction of mitophagy by the mitochondrial uncoupler FCCP is independent of the effect of mitochondrial membrane potential but dependent on acidification of the cytosol by FCCP. The ionophore nigericin also reduces cytosolic pH and induces PINK1/PARKIN-dependent and -independent mitophagy. The increase of intracellular pH with monensin suppresses the effects of FCCP and nigericin on mitochondrial degradation. Thus, a change in intracellular pH is a regulator of mitochondrial quality control. PMID:26893374

  5. The effect of acidic pH on the ability of Clostridium sporogenes MD1 to take up and retain intracellular potassium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    At pH values less 5.5, Clostridium sporogenes MD1 accumulated potassium even though it had little protonmotive force, and an ATPase inhibitor (N, N'- dicyclohexylcarbodiimide) prevented this uptake. These results suggested that potassium transport was ATP-driven, and a protonophore (3, 3', 4', 5 - ...

  6. pH sensing by intracellular Salmonella induces effector translocation.

    PubMed

    Yu, Xiu-Jun; McGourty, Kieran; Liu, Mei; Unsworth, Kate E; Holden, David W

    2010-05-21

    Salmonella enterica is an important intracellular bacterial pathogen of humans and animals. It replicates within host-cell vacuoles by delivering virulence (effector) proteins through a vacuolar membrane pore made by the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS). T3SS assembly follows vacuole acidification, but when bacteria are grown at low pH, effector secretion is negligible. We found that effector secretion was activated at low pH from mutant strains lacking a complex of SPI-2-encoded proteins SsaM, SpiC, and SsaL. Exposure of wild-type bacteria to pH 7.2 after growth at pH 5.0 caused dissociation and degradation of SsaM/SpiC/SsaL complexes and effector secretion. In infected cells, loss of the pH 7.2 signal through acidification of host-cell cytosol prevented complex degradation and effector translocation. Thus, intravacuolar Salmonella senses host cytosolic pH, resulting in the degradation of regulatory complex proteins and effector translocation. PMID:20395475

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

    PubMed

    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. PMID:24256552

  8. /sup 31/P nuclear magnetic resonance measurements of intracellular pH in giant barnacle muscle

    SciTech Connect

    Hamm, J.R.; Yue, G.M.

    1987-01-01

    The accuracy of intracellular pH (pH/sub i/) measurements by /sup 31/P nuclear magnetic resonance (NMR) spectroscopy was examined in single muscle fibers from the giant barnacle, Balanus nubilis. The pH/sub i/ was derived from the chemical shifts of 2-deoxy-D-glucose-6-phosphate and inorganic phosphate. In fibers superfused with sea water at pH 7.7, pH/sub i/ = 7.30 +/- 0.02 at 20/sup 0/C. Experimentally induced pH/sub i/ changes were followed with a time resolution of 3 min. Intracellular alkalinization was induced by exposure to NH/sub 3/Cl and intracellular acidification followed when NH/sub 3/ was removed. Then acid extrusion was stimulated by exposure to bicarbonate containing sea water. In single muscle fibers /sup 31/P NMR results were in excellent agreement with microelectrode studies over the pH range of 6.5 to 8.0. The initial acid extrusion rate was 1.7 +/- 0.3 mmol x 1/sup -1/ x min/sup -1/ at pH/sub i/ 6.75. The authors results showed that /sup 31/P NMR is a reliable in vivo pH probe.

  9. 31P nuclear magnetic resonance measurements of intracellular pH in giant barnacle muscle.

    PubMed

    Hamm, J R; Yue, G M

    1987-01-01

    The accuracy of intracellular pH (pHi) measurements by 31P nuclear magnetic resonance (NMR) spectroscopy was examined in single muscle fibers from the giant barnacle, Balanus nubilis. The pHi was derived from the chemical shifts of 2-deoxy-D-glucose-6-phosphate and inorganic phosphate. In fibers superfused with sea water at pH 7.7, pHi = 7.30 +/- 0.02 at 20 degrees C. Experimentally induced pHi changes were followed with a time resolution of 3 min. Intracellular alkalinization was induced by exposure to NH4Cl and intracellular acidification followed when NH3 was removed. Then acid extrusion was stimulated by exposure to bicarbonate containing sea water. In single muscle fibers 31P NMR results were in excellent agreement with microelectrode studies over the pH range of 6.5 to 8.0. The initial acid extrusion rate was 1.7 +/- 0.3 mmol X l-1 X min-1 at pHi 6.75. Our results showed that 31P NMR is a reliable in vivo pH probe. PMID:3812665

  10. Intracellular and extracellular pH dynamics in the human placenta from diabetes mellitus.

    PubMed

    Araos, Joaquín; Silva, Luis; Salsoso, Rocío; Sáez, Tamara; Barros, Eric; Toledo, Fernando; Gutiérrez, Jaime; Pardo, Fabián; Leiva, Andrea; Sanhueza, Carlos; Sobrevia, Luis

    2016-07-01

    The placenta is a vital organ whose function in diseases of pregnancy is altered, resulting in an abnormal supply of nutrients to the foetus. The lack of placental vasculature homeostasis regulation causes endothelial dysfunction and altered vascular reactivity. The proper distribution of acid- (protons (H(+))) and base-equivalents through the placenta is essential to achieve physiological homeostasis. Several membrane transport mechanisms that control H(+) distribution between the extracellular and intracellular spaces are expressed in the human placenta vascular endothelium and syncytiotrophoblast, including sodium (Na(+))/H(+) exchangers (NHEs). One member of the NHEs family is NHE isoform 1 (NHE1), whose activity results in an alkaline intracellular pH (high intracellular pH (pHi)) and an acidic extracellular pH (pHo). Increased NHE1 expression, maximal transport activity, and turnover are reported in human syncytiotrophoblasts and lymphocytes from patients with diabetes mellitus type I (DMT1), and a positive correlation between NHEs activity and plasma factors, such as that between thrombin and platelet factor 3, has been reported in diabetes mellitus type II (DMT2). However, gestational diabetes mellitus (GDM) could result in a higher sensitivity of the human placenta to acidic pHo. We summarized the findings on pHi and pHo modulation in the human placenta with an emphasis on pregnancies in which the mother diagnosed with diabetes mellitus. A potential role of NHEs, particularly NHE1, is proposed regarding placental dysfunction in DMT1, DMT2, and GDM. PMID:27324099

  11. Intracellular pH and the Control of Multidrug Resistance

    NASA Astrophysics Data System (ADS)

    Simon, Sanford; Roy, Deborshi; Schindler, Melvin

    1994-02-01

    Many anticancer drugs are classified as either weak bases or molecules whose binding to cellular structures is pH dependent. Accumulation of these drugs within tumor cells should be affected by transmembrane pH gradients. Indeed, development of multidrug resistance (MDR) in tumor cells has been correlated with an alkaline shift of cytosolic pH. To examine the role of pH in drug partitioning, the distribution of two drugs, doxorubicin and daunomycin, was monitored in fibroblasts and myeloma cells. In both cell types the drugs rapidly accumulated within the cells. The highest concentrations were measured in the most acidic compartments-e.g., lysosomes. Modifying the cellular pH in drug-sensitive cells to mimic reported shifts in MDR caused an immediate change in the cellular drug concentration. Drug accumulation was enhanced by acidic shifts and reversed by alkaline shifts. All of these effects were rapid and reversible. These results demonstrate that the alkaline shift observed in MDR is sufficient to prevent the accumulation of chemotherapeutic drugs independent of active drug efflux.

  12. Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia).

    PubMed

    Manzl, Claudia; Krumschnabel, Gerhard; Schwarzbaum, Pablo J; Chabicovsky, Monika; Dallinger, Reinhard

    2004-01-01

    The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined. PMID:14695690

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

    PubMed

    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. PMID:26395962

  14. Novel pH-sensitive probes with a ratiometric detection for intracellular pH

    NASA Astrophysics Data System (ADS)

    Ipuy, Martin; Billon, Cyrielle; Micouin, Guillaume; Samarut, Jacques; Andraud, Chantal; Bretonnière, Yann

    2014-08-01

    The development of new pH-sensitive fluorescent probes based on a push-pull architecture is presented with a 2- dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofurane as strong electron acceptor group. With a small structural change, it is possible to obtain a large range of phenolic pKa from 4.8 to 8.6 with some close to neutrality, underlining the role of the electron density modulation on the acidic properties. Remarkable changes in the optical properties (both absorption and fluorescence) were observed as a function of the pH. Ratiometric imaging of intracellular pH was carried out with the most promising probes and highlighted the possibility to distinguish near-neutral minor pH fluctuations in cells.

  15. Acid loading test (pH)

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/003615.htm Acid loading test (pH) To use the sharing features on this page, please enable JavaScript. The acid loading test (pH) measures the ability of the ...

  16. Measurement of Intracellular pH of Skeletal Muscle with pH-sensitive Glass Microelectrodes*

    PubMed Central

    Carter, Norman W.; Rector, Floyd C.; Campion, David S.; Seldin, Donald W.

    1967-01-01

    We used three methods to examine the relationship among intracellular pH, transmembrane potential, and extracellular pH. Single-barreled electrodes permitted the determination of resting potential and intracellular pH with a minimum of cellular injury. Double-barreled electrodes, which incorporated a reference as well as a pH-sensitive electrode in a single tip, facilitated the direct measurement of intracellular pH without the interposition of the transmembrane potential. Triple-barreled electrodes permitted measurement of intracellular pH during the controlled hyperpolarization or depolarization of the cell membrane. The results of all three methods were in close agreement and disclosed that the H+ activity of intracellular and extracellular fluid is in electrochemical equilibrium at any given transmembrane potential. This implies that the determinants of intracellular pH are the transmembrane potential and the blood pH. The actual pH of the normal resting muscle cell is 5.99, as estimated from the normal transmembrane potential and blood pH, or as determined by direct measurements of intracellular pH. PMID:6026098

  17. Intracellular pH: Its role in normal development and teratogenesis

    SciTech Connect

    Duggan, C.A.

    1989-01-01

    Reduction of intracellular pH (pH{sub i}) leading to reduced cell proliferation has been proposed as a mechanism by which acetazolamide induces its teratogenic postaxial limb reduction defect in rodents. In vivo studies measured pH{sub i} using a weak acid and found that pH{sub i} decreased with increasing gestational age during the period of organogenesis in C57 mouse embryos. This decreasing pH{sub i} had a high correlation with the simultaneously occurring decrease in the rate of proliferation determined by {sup 3}H-thymidine incorporation. pH{sub i} or pH was measured for the embryo, embryonic plasma, and extraembryonic fluids following a teratogenic dose of acetazolamide in sensitive C57 and resistant SWV mice. Reduced embryonic pH{sub i} was seen only in the sensitive strain while both strains showed decreased pH values for embryo plasma and extraembryonic fluids, with larger reductions found in the C57 strain. The plasma membrane Na{sup +}/H{sup +} antiporter is known to regulate intracellular pH. Treatment with acetazolamide plus amiloride, and inhibitor of the Na{sup +}/H{sup +} antiporter, resulted in a dramatically increased teratogenic response in C57 embryos and several incidences of the specific limb defect in the resistant SWV embryos. The pH{sub i} and pH effects following the combined drug treatment resulted in larger reductions, the magnitude and duration being greatest in the sensitive strain. The presence of a functional Na{sup +}/H{sup +} antiporter in primary cultures of limb bud mesenchymal cells was documented for both strains of mice using a pH sensitive fluorescent dye. Quantitative studies were done to look for functional differences in the Na{sup +}/H{sup +} antiporter of limb cells from acetazolamide sensitive and resistant embryos.

  18. Light adaptation of invertebrate photoreceptors: influence of intracellular pH buffering capacity.

    PubMed Central

    Bolsover, S R; Brown, J E

    1982-01-01

    1. The possible role of pH changes in mediating light adaptation in Limulus ventral photoreceptor cells was studied by intracellular injection of zwitterionic pH buffers. The intracellular concentration of buffer was estimated by inclusion of a radioactive marker in the injection solution. 2. The light-induced increase of intracellular Ca2+ concentration was monitored by intracellular aequorin. The light-induced increase of Ca2+ concentration was not markedly altered by injection of pH buffer to an intracellular concentration of about 200 mM. 3. The progressive decrease in responsiveness during intracellular ionophoretic injection of Ca2+ was not markedly altered by injection of pH buffer to an intracellular concentration of about 200 mM. 4. Photoreceptors of both Limulus and Balanus were impaled with two micropipettes and voltage clamped. Membrane current induced by a prolonged steady illumination declined from an early transient to a plateau. This delayed decline of current indicates a light-induced reduction of sensitivity (i.e. light adaptation). The wave forms were similar before and after injection of pH buffer to an intracellular concentration of about 200 mM. 5. We conclude that it is unlikely that a light-induced change of cytosolic pH mediates light adaptation in Limulus (and Balanus) photoreceptors. PMID:7175745

  19. Acid Rain, pH & Acidity: A Common Misinterpretation.

    ERIC Educational Resources Information Center

    Clark, David B.; Thompson, Ronald E.

    1989-01-01

    Illustrates the basis for misleading statements about the relationship between pH and acid content in acid rain. Explains why pH cannot be used as a measure of acidity for rain or any other solution. Suggests that teachers present acidity and pH as two separate and distinct concepts. (RT)

  20. A volatile inhibitor immobilizes sea urchin sperm in semen by depressing the intracellular pH

    SciTech Connect

    Johnson, C.H.; Clapper, D.L.; Winkler, M.M.; Lee, H.C.; Epel, D.

    1983-08-01

    Sea urchin spermatozoa are normally immotile in semen, but motility can be initiated by increasing gas flow over the semen--for example, by blowing N2 gas over a thin layer of semen. This result indicates that sperm motility is not O2 limited and suggests that seminal fluid contains a volatile inhibitor of motility which is responsible for the paralysis of sperm in semen. This inhibitor might be carbon dioxide, which reversibly immobilizes sperm. /sup 31/P-NMR measurements of pH show that the sperm intracellular pH (pHi) increases by 0.36 pH unit upon dilution of semen into seawater. Since previous studies have shown that this magnitude of pH increase is sufficient to trigger sperm motility, we suggest that the volatile inhibitor is inhibiting sperm motility in semen by depressing the pHi. A simple hypothesis that explains these observations is that the volatile motility inhibitor is CO/sub 2/, which could acidify pHi as a diffusable weak acid. In this regard, sperm diluted into seawater release acid, and this acid release is related to the pHi increase and motility initiation. In fact, nearly half of the acid released by sperm upon dilution is volatile and may therefore be due to CO/sub 2/ efflux. Most of the acid, however, cannot be attributed to CO/sub 2/ release because it is not volatile. Thus, when sperm are diluted into seawater, they raise their pHi by releasing CO/sub 2/ and protons from the cytoplasm into the surrounding seawater.

  1. The control of intracellular pH in cultured avian chondrocytes.

    PubMed Central

    Dascalu, A; Nevo, Z; Korenstein, R

    1993-01-01

    1. Mechanical loading of cartilaginous tissue generates an increase in the concentration of cations in the extracellular matrix. This includes a decrease of the extracellular pH (pHo), which is known to affect the intracellular pH (pHi), thereby modifying the intracellular metabolism. Thus, the regulation of pHi is essential for the physiological function of cartilage. The fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) was employed in order to assess the mechanisms responsible for control of the pHi in an embryonic avian chondrocyte cell suspension. 2. Steady-state pHi in the absence of physiological HCO3- was 7.15 +/- 0.01 pH units as compared to a pHi of 6.94 +/- 0.02 pH units in its presence (P < 0.01). The intrinsic buffering power of chondrocytes (beta i) was 38.9 mM/pH unit and the total buffering capacity (beta T) was 65.8 mM/pH unit. 3. Cells maintained in a Hepes-buffered solution were exposed to an intracellular acid load by the NH4+ prepulse technique (20 mM NH4Cl). The initial rate of pHi recovery was 0.106 pH units/min (n = 18). Amiloride (0.33 mM), an inhibitor of the Na(+)-H+ exchanger, or replacement of external sodium [Na+]o with choline induced a 60% inhibition of the recovery rate, indicating a predominant involvement of this antiporter in the response to intracellular acidification. 4. H(+)-ATPase inhibitors (oligomycin 20 micrograms/ml; N,N;-dicyclohexylcarbodiimide (DCC), 0.5 mM; N-ethylmaleimide (NEM), 0.25 mM) and iodomycin (2 mM), a metabolic cell suppressor, reduced acid extrusion by 25% as measured by the NH4Cl prepulse in Hepes-bathed cells. 5. Chondrocytes transferred from a Hepes-buffered solution to a 5% CO2-25 mM HCO3- medium (HCO3- solution) underwent a pHi decrease of approximately 0.20 pH units, followed by a regulatory alkalinizing response of 0.118 pH units/min. The Na(+)-H+ exchanger was responsible for only 15% of this alkalinization (amiloride, 0.33 mM), in contrast

  2. Acid loading test (pH)

    MedlinePlus

    The acid loading test (pH) measures the ability of the kidneys to send acid to the urine when there is too much acid in the ... Urine with a pH less than 5.3 is normal. Normal value ranges may vary slightly among different laboratories. Some labs use different ...

  3. Sodium-dependent control of intracellular pH in Purkinje fibres of sheep heart.

    PubMed Central

    Ellis, D; MacLeod, K T

    1985-01-01

    Intracellular pH (pHi) of Purkinje fibres from sheep heart was recorded with pH-sensitive glass micro-electrodes. The cells were acidified by one of three methods: (1) exposure to and subsequent removal of NH4Cl, (2) exposure to solutions containing 5% CO2 or (3) exposure to an acidic Tyrode solution. The pHi recovery from these acidifications was studied. The time constant of recovery from an acidification induced by NH4Cl was almost twice as long as that from one induced by CO2 or acid extracellular pH. Following an acidification induced by exposure to CO2 the time constant of pHi recovery was not changed when the cell was depolarized to -40 mV (by replacement of some Na+ by K+). An intracellular acidification was produced when extracellular Na+ was removed and replaced by quaternary ammonium ions or K+. Such Na+-free solutions also inhibited pHi recovery from an acidification. A 50% inhibition of the rate of recovery was produced by lowering the [Na+]o to 8 mM. When used as a Na+ substitute, Li+ could permit recovery. Tris (22 mM) changed pHi in the alkaline direction. Amiloride (1 mM) or a decrease in temperature slowed the recovery from an acidification (Q10 = 2.65). There was no effect of SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid disodium salt; 100 microM) on the recovery. Na+-sensitive glass micro-electrodes were used to measure the intracellular Na+ activity when [Na+]o was lowered to levels used in our pHi recovery experiments. From these data we have calculated the apparent Na+ electrochemical gradient at different values of [Na+]o. If this gradient is responsible for H+ efflux from the cell then, by applying thermodynamic considerations, it can be shown that only low concentrations (1-2mM) of extracellular Na+ are required. Solutions containing a very low [Ca2+]o (less than 10(-8) M, buffered with EGTA) were used to prevent large rises of [Ca2+]i which may occur on removal of external Na+. Under these conditions pHi recovery is

  4. Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia.

    PubMed

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

    2013-04-01

    Acid-extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid-extrusion remains unclear. We studied pH-regulation under normoxia and hypoxia in eight cancer cell-lines (HCT116, RT112, MDA-MB-468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH-sensitive fluorophore, cSNARF-1. Hypoxia responses were triggered by pre-incubation in low O(2) or with the 2-oxoglutarate-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport-substrate removal, acid-extrusion flux was dissected into components due to Na(+)/H(+) exchange (NHE) and Na(+)-dependent HCO(3)(-) transport. In half of the cell-lines (HCT116, RT112, MDA-MB-468, MCF10A), acid-extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA-MB-468), NHE-flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4-h hypoxic incubation reduced NHE-flux reversibly with a time-constant of 1-2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48-h hypoxia, inhibition of NHE-flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid-extrusion by Na(+)-dependent HCO(3)(-) transport was hypoxia-insensitive and comparable in all cell lines. This constitutive and stable element of pH-regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell-specific differences in their dynamic response to larger acid loads. PMID:22949268

  5. Application of SERS Nanoparticles for Intracellular pH Measurements

    SciTech Connect

    Laurence, T; Talley, C; Colvin, M; Huser, T

    2004-10-21

    We present an alternative approach to optical probes that will ultimately allow us to measure chemical concentrations in microenvironments within cells and tissues. This approach is based on monitoring the surface-enhanced Raman scattering (SERS) response of functionalized metal nanoparticles (50-100 nm in diameter). SERS allows for the sensitive detection of changes in the state of chemical groups attached to individual nanoparticles and small clusters. Here, we present the development of a nanoscale pH meter. The pH response of these nanoprobes is tested in a cell-free medium, measuring the pH of the solution immediately surrounding the nanoparticles. Heterogeneities in the SERS signal, which can result from the formation of small nanoparticle clusters, are characterized using SERS correlation spectroscopy and single particle/cluster SERS spectroscopy. The response of the nanoscale pH meters is tested under a wide range of conditions to approach the complex environment encountered inside living cells and to optimize probe performance.

  6. Agonist-mediated changes in intracellular pH: role in vascular smooth muscle cell function

    SciTech Connect

    Berk, B.C.; Canessa, M.; Vallega, G.; Alexander, R.W.

    1988-01-01

    Changes in intracellular pH (pHi) are likely to play an important role in regulation of vascular smooth muscle cell (VSMC) function. In most blood vessels, acidification is associated with decreased contractile tone and alkalinization with increased tone. However, the nature of agonist-mediated alterations in pHi and the role of pHi in other VSMC responses has been little studied. We have used the pH sensitive dye, BCECF, to study pHi in cultured rat aortic VSMC. Basal pHi at 37 degrees C in physiologic saline buffer (pH 7.3) was 7.08 in suspended VSMC and 7.26 in substrate-attached VSMC. An amiloride-sensitive Na+/H+ exchanger mediated pHi recovery following an acid load. Angiotensin II- and platelet-derived growth factor typified one class of VSMC agonists, causing an initial transient (less than 5 min) acidification followed by a sustained (greater than 20 min) alkalinization. The acidification phase was associated with increased Ca2+ mobilization as demonstrated by increases in intracellular Ca2+ and 45Ca2+ efflux. The alkalinization was associated with Na+ influx and H+ efflux consistent with Na+/H+ exchange. Epidermal growth factor and phorbol esters typified another class of agonists which stimulated only a sustained alkalinization. Alterations in regulation of VSMC pHi may play an important role in VSMC hypertrophy and/or proliferation as suggested by the finding of increased cell growth and Na+/H+ exchange in spontaneously hypertensive rat VSMC compared to Wistar-Kyoto VSMC. Although no functional correlate for initial acidification has been identified, cytoplasmic alkalinization appears to be required for the sustained formation of diacylglycerol following angiotensin II stimulation. These findings suggest that alterations in pHi may regulate several VSMC functions such as agonist-mediated signal transduction, excitation-response coupling, and growth.

  7. Intracellular pH of perfused single frog skin: combined 19F- and 31P-NMR analysis.

    PubMed

    Civan, M M; Lin, L E; Peterson-Yantorno, K; Taylor, J; Deutsch, C

    1984-11-01

    Intracellular pH (pHc) has been determined in frog skin by applying two different methods of pH measurement, 19F and 31P nuclear magnetic resonance (NMR) analysis, to the same tissues. Results from both NMR approaches confirm an observation by Lin, Shporer, and Civan [Am. J. Physiol. 248 (Cell Physiol. 17): 1985] that acidification of the extracellular medium reverses the sign of the pH gradient present under baseline conditions. The fluorinated probe, alpha-(difluoromethyl)-alanine methyl ester, was introduced into the epithelial cells by preincubating skins for 4.7-10.4 h at room temperature in Ringer solutions containing 1 mM ester. The free amino acid was subsequently released by intracellular esterase activity, thus providing a high enough probe concentration for NMR analysis to be practicable. From measurements of short-circuit current and transepithelial resistance under base-line and experimental conditions and the appearance of phosphocreatine (PCr) in the 31P spectrum of preloaded tissues, the fluorinated probe appears to be nontoxic to frog skin. Measurement of the chemical shift of methylphosphonate relative to PCr permitted calculation of extracellular pH. Estimation of the intracellular pH was performed both by measurement of the chemical shift of inorganic phosphate (Pi) relative to PCr and by measurement of the central peak spacing of the 19F spectrum. From four direct comparisons of the two techniques in two experiments, the difference in the estimated pH was only 0.03 +/- 0.07 pH units, supporting the concept that 31P-NMR analysis is a valid method of measuring pH in this tissue. PMID:6496729

  8. Intracellular pH regulation in isolated rat bile duct epithelial cells.

    PubMed Central

    Strazzabosco, M; Mennone, A; Boyer, J L

    1991-01-01

    To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport. Images PMID:2022723

  9. Cell-specific intracellular anticancer drug delivery from mesoporous silica nanoparticles with pH sensitivity.

    PubMed

    Luo, Zhong; Cai, Kaiyong; Hu, Yan; Zhang, Beilu; Xu, Dawei

    2012-05-01

    A nanoreservoir for efficient intracellular anticancer drug delivery based on mesoporous silica nanoparticles end-capped with lactobionic acid-grafted bovine serum albumin is fabricated. It demonstrates great potential for both cell-specific endocytosis and intracellular pH-responsive controlled release of drugs. A possible endocytosis pathway/mechanism of the smart controlled drug release system is proposed. PMID:23184747

  10. Quantitative modelling of the effects of selected intracellular metabolites on pH in fish white muscle

    PubMed

    Arthur; West; Hochachka

    1997-01-01

    A model is presented that provides guidelines to the identification of key experimental variables influencing proton balance and intracellular pH in vertebrate white muscle. We have drawn on data from the literature on rainbow trout (Oncorhynchus mykiss) in an attempt to quantify the influence of metabolic, ionic and transport components of proton generation and proton consumption after exercise. Only minor changes in proton balance and in calculated intracellular pH were caused by considering changes in the concentration of bicarbonate or including the acid­base characteristics of purine nucleotides. Intracellular pH, as estimated by the model, was more acidic at some time points in recovery compared with in vivo measurements, and this would appear to result mainly from inaccuracies in quantifying the phosphate component of proton buffering. Nevertheless, the model was able to simulate the typical pattern of muscle acidosis and recovery observed for trout, including the transient post-exercise acidification and the slow recovery rate. As with previous pHi models, comparison of model estimates with experimental observations is essential in this approach in order to identify whether all of the relevant metabolic processes have been considered for accurate quantification of proton balance within the white muscle compartment. PMID:9319037

  11. Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

    SciTech Connect

    Bond, P.L.; Keller, J.; Blackall, L.L.

    1999-06-05

    The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. The authors investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. The authors observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. from these observations, the authors postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimization of EBPR.

  12. Construction of pH-Sensitive "Submarine" Based on Gold Nanoparticles with Double Insurance for Intracellular pH Mapping, Quantifying of Whole Cells and in Vivo Applications.

    PubMed

    Yu, Kang-Kang; Li, Kun; Qin, Hui-Huan; Zhou, Qian; Qian, Chen-Hui; Liu, Yan-Hong; Yu, Xiao-Qi

    2016-09-01

    A series of "submarines", which composed of gold nanoparticles and modified with rhodamine and fluorescein derivatives, were presented. With dual sensitive units for both acidic and basic environment, these "gold nano-submarines" not only allow efficient intracellular pH mapping but also provide more accurate quantitative detection of pH alteration under different stimuli with distinct pH quantification range. Moreover, they even have the ability to pass through the blood brain barrier (BBB). PMID:27532147

  13. A two-photon ratiometric fluorescent probe enables spatial coordinates determination of intracellular pH.

    PubMed

    Wang, Junjie; Sun, Yuming; Zhang, Weijia; Liu, Yong; Yu, Xiaoqiang; Zhao, Ning

    2014-11-01

    We reported a two-photon ratiometric fluorescent probe for detecting intracellular pH. When excited with 800 nm laser, an optimal output of laser as the routine equipment of two-photon fluorescence microscopy, the two-photon excited fluorescence of this probe showed distinct emission peak shift as large as 109 nm upon the change of pH values in vitro. Very importantly, the experiment results show that this probe has large two-photon absorption cross-section at pH 4.5 at 800 nm of 354 g, which ranks it as one of the best two-photon ratiometric fluorescent pH probes, and its working pH value is between 4.0 and 8.0 which could fit the intracellular pH range. Moreover, utilizing this probe, the two-photon ratiometric fluorescent images in living cells have been obtained, and the spatial coordinates of intracellular pH can be mapped. At the same time, the probe also exhibited selectivity, photostability and membrane permeability. And the photophysical properties of this probe in various solvents indicated that these photophysical properties variations are due to an intramolecular charge transfer process. At last, the imaging depth of the probe in liver biopsy slices was investigated. The experimental results demonstrated the maximum imaging depth can arrive 66 µm in living rat liver tissues. PMID:25127590

  14. Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells

    PubMed Central

    Taylor, Caroline J; Nicola, Pieris A; Wang, Shanshan; Barrand, Margery A; Hladky, Stephen B

    2006-01-01

    Fluid secretion across the blood–brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO3−, which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pHi). To analyse the transporters that may be involved in the maintenance of pHi and hence secretion of HCO3−, we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO2/HCO3−. pHi in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO3− than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO3− were similar. In the nominal absence of HCO3− the rate of acid loading was increased equally by removal of external Na+ or by inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO3− the increase in the rate of acid loading when Na+ was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na+ and HCO3−. Removal of Cl− in the presence of HCO3− produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na+ following removal of Cl− increased the rate of acid gain. In the presence of HCO3− and initial presence of Na+ and Cl−, DIDS inhibited the changes in pHi produced by removal of either Na+ or Cl−. These are the expected results if these cells possess an AE-like Cl−/HCO3− exchanger, a ‘channel-like’ permeability allowing slow influx of acid (or efflux of HCO3−), a NBC-like Cl−-independent Na+−HCO3− cotransporter, and a NHE-like Na+/H+ exchanger. The in vitro rates of HCO3− loading via the Na+−HCO3

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

    PubMed Central

    Naz, Rajesh K.

    2014-01-01

    Background 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. Methods 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)]. Results Curcumin caused a concentration-dependent (p<0.05) decrease in forward motility of both human and mouse sperm. It also caused a concentration-dependent decrease in intracellular pH (pHi) in both human and mouse sperm. Curcumin induced significant (p<0.05) hyperpolarization of the plasma membrane in both human and mouse sperm. Conclusion These findings indicate that curcumin inhibits sperm forward motility by intracellular acidification and hyperpolarization of sperm plasma membrane. This is the first study to our knowledge which examined the effect of curcumin on sperm pHi 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. PMID:24918078

  16. Intracellular pH regulation in resting and contracting segments of rat mesenteric resistance vessels.

    PubMed Central

    Aalkjaer, C; Cragoe, E J

    1988-01-01

    1. The pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF) was used to measure intracellular pH (pHi) in segments of rat resistance vessels (internal diameter about 200 microns) with the vessels mounted in a myograph for simultaneous measurements of isometric contraction. 2. BCECF loaded slowly into the vessels over 1 h and did not affect the maximal contractility of the vessels. There was a loss of dye with time which, however, was very slow when the segments were only excited for 2 s/min, suggesting that the loss was mainly due to dye bleaching with only a very slow leak. 3. The ratio of the emissions (at 540 nm) with excitation at 495 and 450 nm was calibrated in terms of pH using the K+-H+ ionophore nigericin. This calibration gave a pHi value of 7.15 +/- 0.02 (n = 20), suggesting that hydrogen ions are not in electrochemical equilibrium in these vascular smooth muscles which have a membrane potential of about -60 mV. 4. Addition of 10 mM-NH4Cl caused a transient alkalinization and wash-out of 10 mM-NH4Cl a transient acidification. Increasing CO2 with maintained bicarbonate caused a rapid acidification followed by an incomplete recovery. Removal of CO2 and bicarbonate (HEPES-buffered solution) with constant extracellular pH caused a transient alkalinization but steady-state pHi was not significantly altered. 5. In bicarbonate-free buffer the Na+-H+ exchange blocker 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and sodium-free conditions caused a slow acidification. In bicarbonate buffer (PSS) EIPA had no detectable effect after 10 min but the anion exchange blocker diisothio-cyanatostilbenedisulphonic acid (DIDS) caused a small acidification over that time course. 6. The rate of recovery after an acid load was about 50% lower in HEPES buffer compared to PSS and it was inhibited by EIPA. In PSS amiloride and EIPA each had a small inhibitory effect on the pH recovery after an acid load. DIDS also inhibited the recovery from an acid load

  17. Influence of extracellular pH on growth, viability, cell size, acidification activity, and intracellular pH of Lactococcus lactis in batch fermentations.

    PubMed

    Hansen, Gunda; Johansen, Claus Lindvald; Marten, Gunvor; Wilmes, Jacqueline; Jespersen, Lene; Arneborg, Nils

    2016-07-01

    In this study, we investigated the influence of three extracellular pH (pHex) values (i.e., 5.5, 6.5, and 7.5) on the growth, viability, cell size, acidification activity in milk, and intracellular pH (pHi) of Lactococcus lactis subsp. lactis DGCC1212 during pH-controlled batch fermentations. A universal parameter (e.g., linked to pHi) for the description or prediction of viability, specific acidification activity, or growth behavior at a given pHex was not identified. We found viability as determined by flow cytometry to remain high during all growth phases and irrespectively of the pH set point. Furthermore, regardless of the pHex, the acidification activity per cell decreased over time which seemed to be linked to cell shrinkage. Flow cytometric pHi determination demonstrated an increase of the averaged pHi level for higher pH set points, while the pH gradient (pHi-pHex) and the extent of pHi heterogeneity decreased. Cells maintained positive pH gradients at a low pHex of 5.5 and even during substrate limitation at the more widely used pHex 6.5. Moreover, the strain proved able to grow despite small negative or even absent pH gradients at a high pHex of 7.5. The larger pHi heterogeneity at pHex 5.5 and 6.5 was associated with more stressful conditions resulting, e.g., from higher concentrations of non-dissociated lactic acid, while the low pHi heterogeneity at pHex 7.5 most probably corresponded to lower concentrations of non-dissociated lactic acid which facilitated the cells to reach the highest maximum active cell counts of the three pH set points. PMID:27020293

  18. Rationally Engineering Phototherapy Modules of Eosin-Conjugated Responsive Polymeric Nanocarriers via Intracellular Endocytic pH Gradients.

    PubMed

    Liu, Guhuan; Hu, Jinming; Zhang, Guoying; Liu, Shiyong

    2015-07-15

    Spatiotemporal switching of respective phototherapy modes at the cellular level with minimum side effects and high therapeutic efficacy is a major challenge for cancer phototherapy. Herein we demonstrate how to address this issue by employing photosensitizer-conjugated pH-responsive block copolymers in combination with intracellular endocytic pH gradients. At neutral pH corresponding to extracellular and cytosol milieu, the copolymers self-assemble into micelles with prominently quenched fluorescence emission and low (1)O2 generation capability, favoring a highly efficient photothermal module. Under mildly acidic pH associated with endolysosomes, protonation-triggered micelle-to-unimer transition results in recovered emission and enhanced photodynamic (1)O2 efficiency, which synergistically actuates release of encapsulated drugs, endosomal escape, and photochemical internalization processes. PMID:25514473

  19. The amiloride-sensitive Na+/H+ exchange antiporter and control of intracellular pH in hippocampal brain slices.

    PubMed

    Lin, C W; Kalaria, R N; Kroon, S N; Bae, J Y; Sayre, L M; LaManna, J C

    1996-08-26

    The intracellular pH, 7.54 +/- 0.03 (mean +/- S.D., n = 15), determined with the Neutral red method, of the hippocampal brain slice preparation under baseline incubation conditions is considerably more alkaline than the bath buffer pH. Neutralization by amiloride suggests that the alkalinity was due to Na+/H+ exchange antiporter activation. To characterize the brain Na+/H+ exchange antiporter we compared the inhibitory effects of MIA, amiloride and other 5-N substituted analogues on proton extrusion after acid loading by transient exposure to ammonium chloride in the isolated hippocampal brain slice preparation. The potencies of amiloride compounds on the initial recovery rate of intracellular pH after acid-loading were DMA > MIA > HMA = MHA > or = IPA-HCI > IPA > MNPA = Amil > Benzamil. The greater potency of the 5-N substituted analogs of amiloride over amiloride and benzamil strongly suggest that Na+/H+ exchange antiporter is the mechanism responsible for alkalinization in the isolated hippocampal brain slice in vitro. PMID:8883860

  20. Intracellular pH and ionic channels in the Loligo vulgaris giant axon.

    PubMed Central

    Carbone, E; Testa, P L; Wanke, E

    1981-01-01

    Squid giant axons were used to investigate the reversible effects of intracellular pH(pHi) on the kinetic properties of ionic channels. The pharmacologically separated K+ and Na+ currents were measured under: (a) internal perfusion, (b) enzymatic Pronase treatment, and (c) continuous estimate of periaxonal ion accumulation. Variation of internal pH from 4.8 to 11 resulted in: (a) a decrease of steady-state sodium inactivation at positive potentials similar to the effect of the proteolytic enzyme Pronase, (b) a shift of the h infinity (E) curve toward depolarizing voltages, and (c) a decrease of the time constant of inactivation for potentials below -20 mV (an increase above). A plot of the steady-state sodium conductance at E = +40 mV as a function of pHi suggests that two groups with pKa 10.4 and 5.6 affect respectively the inactivation gate and the rate constants for the transition from the inactivated to the second open state (h2) (Chandler and Meves, 1970b). The voltage shifts of the kinetic parameters predicted by the Gouy-Chapman-Stern theory are well satisfied at high pHi and less at low. Once corrected for voltage shifts, the forward rate constants for channel opening were found to be slowed with the acidity of the internal or external solution. PMID:6268214

  1. EFFECT OF TETRACYCLINES ON THE INTRACELLULAR AMINO ACIDS OF MOLDS.

    PubMed

    FREEMAN, B A; CIRCO, R

    1963-07-01

    Freeman, Bob A. (University of Chicago, Chicago, Ill.) and Richard Circo. Effect of tetracyclines on the intracellular amino acids of molds. J. Bacteriol. 86:38-44. 1963.-The tetracycline antibiotics were shown to alter the amino acid metabolism of molds whose growth is not markedly affected. Eight molds were grown in the presence of these antiobiotics; four exhibited a general reduction in the concentration of the intracellular amino acids, except for glutamic acid and alanine. In most of these four cultures, the tetracyclines also caused the complete disappearance of arginine, lysine, proline, phenylalanine, and tyrosine from the intracellular amino acid pool. The significance of these observations and the usefulness of the method in the study of the mechanisms of antibiotic action are discussed. PMID:14051820

  2. The biophysical and molecular basis of intracellular pH sensing by Na+/H+ exchanger-3

    PubMed Central

    Babich, Victor; Vadnagara, Komal; Di Sole, Francesca

    2013-01-01

    Epithelial Na+/H+ exchanger-3 (NHE3) transport is fundamental for renal and intestinal sodium reabsorption. Cytoplasmic protons are thought to serve as allosteric modifiers of the exchanger and to trigger its transport through protein conformational change. This effect presupposes an intracellular pH (pHi) dependence of NHE3 activity, although the biophysical and molecular basis of NHE3 pHi sensitivity have not been defined. NHE3, when complexed with the calcineurin homologous protein-1 (CHP1), had a shift in pHi sensitivity (0.4 units) toward the acidic side in comparison with NHE3 alone, as measured by oscillating pH electrodes combined with whole-cell patch clamping. Indeed, CHP1 interaction with NHE3 inhibited NHE3 transport in a pHi -dependent manner. CHP1 binding to NHE3 also affected its acute regulation. Intracellular perfusion of peptide from the CHP1 binding region (or pHi modification to reduce the CHP1 amount bound to NHE3) was permissive and cooperative for dopamine inhibition of NHE3 but reversed that of adenosine. Thus, CHP1 interaction with NHE3 apparently establishes the exchanger set point for pHi, and modification in this set point is effective in the hormonal stimuli–mediated regulation of NHE3. CHP1 may serve as a regulatory cofactor for NHE3 conformational change, dependent on intracellular protonation.—Babich V., Vadnagara K., Di Sole, F. The biophysical and molecular basis of intracellular pH sensing by the Na+/H+ exchanger-3. PMID:23934281

  3. The temperature dependence of intracellular pH in isolated frog skeletal muscle: lessons concerning the Na(+)-H+ exchanger.

    PubMed

    Marjanovic, M; Elliott, A C; Dawson, M J

    1998-02-01

    We used 31P NMR to investigate the temperature-dependence of intracellular pH (pHi) in isolated frog skeletal muscles. We found that Ln[H+i] is a linear function of 1/Tabs paralleling those of neutral water (i.e., H+ = OH-) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na(+)-H+ exchange shifted the intercept in the alkaline direction but had not effect on the slope. Acid loading followed by washout resulted in an amiloride-sensitive return to the (temperature dependent) basal pHi. These results show that the temperature dependence of activation of Na(+)-H+ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H+]/ [OH-] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point of the Na(+)-H+ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (delta H degree) under both control and insulin-stimulated conditions that is similar to the delta H degree of the intracellular buffers and approximately half of the delta H degree for the dissociation of water. Thus, the temperature-dependent component of the standard free-energy change (delta F degree) is unaffected by insulin stimulation, suggesting that changes in Arrhenius activation energy (Ea) may not be a part of the mechanism of hormone stimulation. PMID:9493127

  4. Nuclear magnetic resonance studies of intracellular pH and pH homeostasis in the hog carotid artery

    SciTech Connect

    Grieder, T.A.

    1989-01-01

    Intracellular pH (pH{sub i}) is an important determinant of vascular smooth muscle (VSM) contractility and relaxation. Most NMR measurement of pH have been calculated from the chemical shift of inorganic phosphate (P{sub i}) in {sup 31}P spectra. An alternative approach is to calculate pH from the difference in chemical shifts of signals in the {sup 19}F spectrum of cells loaded with difluoromethylalanine. This technique has higher sensitivity to pH changes and provides better time resolution than other NMR methods. In this study we report simultaneous measurements of pH{sub i} and the contractile state of single, intact hog carotid arterial segments, closed at both ends and superfused with HCO{sub 3}{sup {minus}}-buffered Krebs solution at physiological pressures. At 28{degree}C, resting arteries maintained a pH{sub i} of 7.15 {+-} 0.03 units (n = 16). In a parallel study, helically cut strips studied with {sup 31}P NMR maintained a similar resting pH (7.18 {+-} 0.09).

  5. Effect of altitude on brain intracellular pH and inorganic phosphate levels

    PubMed Central

    Shi, Xian-Feng; Carlson, Paul J.; Kim, Tae-Suk; Sung, Young-Hoon; Hellem, Tracy L.; Fiedler, Kristen K.; Kim, Seong-Eun; Glaeser, Breanna; Wang, Kristina; Zuo, Chun S.; Jeong, Eun-Kee; Renshaw, Perry F.; Kondo, Douglas G.

    2015-01-01

    Normal brain activity is associated with task-related pH changes. Although central nervous system syndromes associated with significant acidosis and alkalosis are well understood, the effects of less dramatic and chronic changes in brain pH are uncertain. One environmental factor known to alter brain pH is the extreme, acute change in altitude encountered by mountaineers. However, the effect of long-term exposure to moderate altitude has not been studied. The aim of this two-site study was to measure brain intracellular pH and phosphate-bearing metabolite levels at two altitudes in healthy volunteers, using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Increased brain pH and reduced inorganic phosphate (Pi) levels were found in healthy subjects who were long-term residents of Salt Lake City, UT (4720 ft/1438 m), compared with residents of Belmont, MA (20 ft/6 m). Brain intracellular pH at the altitude of 4720 ft was more alkaline than that observed near sea level. In addition, the ratio of inorganic phosphate to total phosphate signal also shifted toward lower values in the Salt Lake City region compared with the Belmont area. These results suggest that long-term residence at moderate altitude is associated with brain chemical changes. PMID:24768210

  6. Effect of cationic side-chains on intracellular delivery and cytotoxicity of pH sensitive polymer-doxorubicin nanocarriers

    NASA Astrophysics Data System (ADS)

    Fang, Chen; Kievit, Forrest M.; Cho, Yong-Chan; Mok, Hyejung; Press, Oliver W.; Zhang, Miqin

    2012-10-01

    Fine-tuning the design of polymer-doxorubicin conjugates permits optimization of an efficient nanocarrier to greatly increase intracellular uptake and cytotoxicity. Here, we report synthesis of a family of self-assembled polymer-doxorubicin nanoparticles and an evaluation of the effects of various types of side-chains on intracellular uptake and cytotoxicity of the nanocarriers for lymphoma cells. Monomers with three different cationic side-chains (CA) and pKa's, i.e., a guanidinium group (Ag), an imidazole group (Im), and a tertiary amine group (Dm), were comparatively investigated. The cationic monomer, poly(ethylene glycol) (PEG), and doxorubicin (Dox) were reacted with 1,4-(butanediol) diacrylate (BUDA) to prepare a poly(β-amino ester) (PBAE) polymer via Michael addition. All three polymer-Dox conjugates spontaneously formed nanoparticles (NP) through hydrophobic interactions between doxorubicin in aqueous solution, resulting in NP-Im/Dox, NP-Ag/Dox, and NP-Dm/Dox, with hydrodynamic sizes below 80 nm. Doxorubicin was linked to all 3 types of NPs with a hydrazone bond to assure selective release of doxorubicin only at acidic pH, as it occurs in the tumor microenvironment. Both NP-Im/Dox and NP-Ag/Dox exhibited much higher intracellular uptake by Ramos cells (Burkitt's lymphoma) than NP-Dm/Dox, suggesting that the type of side chain in the NPs determines the extent of intracellular uptake. As a result, NP-Im/Dox and NP-Ag/Dox showed cytotoxicity that was comparable to free Dox in vitro. Our findings suggest that the nature of surface cationic group on nanocarriers may profoundly influence their intracellular trafficking and resulting therapeutic efficacy. Thus, it is a crucial factor to be considered in the design of novel carriers for intracellular drug delivery.

  7. Uric acid utilization by Mycobacterium intracellulare and Mycobacterium scrofulaceum isolates.

    PubMed Central

    Falkinham, J O; George, K L; Parker, B C; Gruft, H

    1983-01-01

    Forty-nine human and environmental isolates of Mycobacterium intracellulare and Mycobacterium scrofulaceum were tested for their ability to grow on uric acid and a number of its degradation products. Nearly all (88 to 90%) strains used uric acid or allantoin as a sole nitrogen source; fewer (47 to 69%) used allantoate, urea, or possibly ureidoglycollate. Enzymatic activities of one representative isolate demonstrated the existence of a uric acid degradation pathway resembling that in other aerobic microorganisms. PMID:6863220

  8. Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

    PubMed Central

    Alvaro, D; Della Guardia, P; Bini, A; Gigliozzi, A; Furfaro, S; La Rosa, T; Piat, C; Capocaccia, L

    1995-01-01

    To elucidate mechanisms of glucagon-induced bicarbonate-rich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pHi) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pHi, significantly stimulates the Cl-/HCO3- exchange activity. The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3- exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3- exchange was individually blocked by two specific and selective inhibitors of protein kinase A, Rp-cAMPs (10 microM) and H-89 (30 microM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl- channel blocker, NPPB (10 microM), showed no effect on either the basal or the glucagon-stimulated Cl-/HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 microM), completely blocked the glucagon stimulation of the Cl-/HCO3- exchange; however, this effect was achieved through a significant inhibition of the glucagon-stimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3- exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pHi or at acid pHi values. In contrast, glucagon, at basal pHi, stimulated the Na(+)-HCO3- symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3- exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis. Images PMID:7635959

  9. In situ probing of intracellular pH by fluorescence from inorganic nanoparticles.

    PubMed

    Guo, Junhong; Xiong, Shijie; Wu, Xinglong; Shen, Jiancang; Chu, Paul K

    2013-12-01

    Intracellular pH (pHi) plays a critical role in the physiological processes of cells. Nanoscale sensors based on pH-sensitive fluorescent proteins attached on nanoparticles (NPs) have been designed but inorganic NP-dependent fluorescent nanosensors have not yet been explored. Herein we describe a pH sensitive inorganic semiconductor fluorescent probe based on ultrathin 3C-SiC NPs which can effectively monitor pH in the range of 5.6-7.4 by taking advantage of the linear dependence between the fluorescent intensity ratio of the surface OH(-) and H(+) bonding states to band-to-band recombination and pH. Detection of pHi is demonstrated in living HeLa cells. In particular, pHi measurements during apoptosis confirm the validity and sensitivity of this technique in monitoring real-time changes in the intracellular environment. Toxicity assessment and confocal laser scanning microscopy indicate that the 3C-SiC NPs have low cytotoxicity and are compatible with living cells. PMID:24008041

  10. Synchronous Bioimaging of Intracellular pH and Chloride Based on LSS Fluorescent Protein.

    PubMed

    Paredes, Jose M; Idilli, Aurora I; Mariotti, Letizia; Losi, Gabriele; Arslanbaeva, Lyaysan R; Sato, Sebastian Sulis; Artoni, Pietro; Szczurkowska, Joanna; Cancedda, Laura; Ratto, Gian Michele; Carmignoto, Giorgio; Arosio, Daniele

    2016-06-17

    Ion homeostasis regulates critical physiological processes in the living cell. Intracellular chloride concentration not only contributes in setting the membrane potential of quiescent cells but it also plays a role in modulating the dynamic voltage changes during network activity. Dynamic chloride imaging demands new tools, allowing faster acquisition rates and correct accounting of concomitant pH changes. Joining a long-Stokes-shift red-fluorescent protein to a GFP variant with high sensitivity to pH and chloride, we obtained LSSmClopHensor, a genetically encoded fluorescent biosensor optimized for the simultaneous chloride and pH imaging and requiring only two excitation wavelengths (458 and 488 nm). LSSmClopHensor allowed us to monitor the dynamic changes of intracellular pH and chloride concentration during seizure like discharges in neocortical brain slices. Only cells with tightly controlled resting potential revealed a narrow distribution of chloride concentration peaking at about 5 and 8 mM, in neocortical neurons and SK-N-SH cells, respectively. We thus showed that LSSmClopHensor represents a new versatile tool for studying the dynamics of chloride and proton concentration in living systems. PMID:27031242

  11. Intracellular pH of brown adipose tissue increases during norepinephrine stimulation of thermogenesis

    SciTech Connect

    Horwitz, B.A.; Hamilton, J.S.

    1986-03-01

    Norepinephrine (NE) activation of brown fat (BAT) thermogenesis appears to involve dissociation of purine nucleotides from the mitochondrial uncoupling protein, resulting in release of normal respiratory control and enhanced substrate oxidation. Since the affinity of the uncoupling protein for purine nucleotides decreases significantly with increasing pH, the authors wished to determine if NE administration shifted the intracellular pH of BAT. To examine this question under in vivo conditions, they positioned a nuclear magnetic resonance (NMR) surface coil over the interscapular BAT of anesthetized male Syrian hamsters. The underlying and surrounding musculature was shielded to minimize their contribution to the /sup 31/P spectra. The hamster was placed in a Nicolet 200 Mhz spectrometer, operating in the Fourier Transform mode and tuned to /sup 31/P. Scans taken during infusion of ascorbate buffer (vehicle for NE) were compared to those taken during NE infusion (8 ng/g x min). During this infusion, BAT temperature increased 3.7 +/- 0.5/sup 0/C, confirming that BAT thermogenesis was activated. There also occurred a statistically significant PPM (parts per million) shift, averaging 0.070 +/- 0.022 (n = 22) and corresponding to an increase of approximately 0.07 pH units. This shift in intracellular pH from 7.32 to 7.39, although small, would facilitate the maintenance of loosely coupled brown fat mitochondria.

  12. Monitoring the intracellular pH of Zygosaccharomyces bailii by green fluorescent protein.

    PubMed

    Dang, T D T; De Maeseneire, S L; Zhang, B Y; De Vos, W H; Rajkovic, A; Vermeulen, A; Van Impe, J F; Devlieghere, F

    2012-06-01

    It is generally known that intracellular pH (pH(i)) plays a vital role in cell physiology and that pH(i) homeostasis is essential for normal cellular functions. Therefore, it is desirable to know the pH(i) during cell life cycle or under various growth conditions. Different methods to measure pH(i) have been developed and among these methods, the use of pH-sensitive green fluorescent protein (GFP) as a pH(i) indicator is a promising technique. By using this approach, not only can more accurate pH(i) results be obtained but also long-term experiments on pH(i) can be performed. In this study, the wild type Zygosaccharomyces bailii, a notorious food spoilage yeast, was transformed with a plasmid encoding a pH-sensitive GFP (i.e. pHluorin), enabling the pH(i) of the yeast to be determined based on cellular fluorescent signals. After the transformation, growth and pH(i) of the yeast were investigated in four different acidic conditions at 22°C during 26days. From the experimental results, the transformation effectiveness was verified and a good correlation between yeast growth and pH(i) was noticed. Particularly, it was observed that the yeast has an ability to tolerate a significant pH(i) drop during exponential phase and a subsequent pH(i) recovery in stationary phase, which may underlie the exceptional acid resistance of the yeast. This was the first time that a GFP-based approach for pH(i) measurement was applied in Z. bailii and that the pH(i) of the yeast was monitored during such a long period (26days). It can be expected that greater understanding of the physiological properties and mechanisms behind the special acid resistance of the yeast will be obtained from further studies on this new yeast strain. PMID:22538167

  13. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment. PMID:27480055

  14. A rapid method for measuring intracellular pH using BCECF-AM.

    PubMed

    Ozkan, Pinar; Mutharasan, Raj

    2002-08-15

    A rapid intracellular pH (pH(i)) measurement method based on initial rate of increase of fluorescence ratio of 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein upon dye addition to a cell suspension in growth medium is reported. A dye transport model that describes dye concentration and fluorescence values in intracellular and extracellular spaces provides the mathematical basis for the approach. Experimental results of ammonium chloride challenge response of the two suspension cells, Spodoptera frugiperda and Chinese hamster ovary (CHO) cells, successfully compared with results obtained using traditional perfusion method. Since the cell suspension does not require any preparation, measurement of pH(i) can be completed in about 1 min minimizing any potential errors due to dye leakage. PMID:12204343

  15. Effect of systemic pH on pH sub i and lactic acid generation in exhaustive forearm exercise

    SciTech Connect

    Hood, V.L.; Schubert, C.; Keller, U.; Mueller, S. Univ. of Vermont College of Medicine, Burlington )

    1988-09-01

    To investigate whether changes in systemic pH affect intracellular pH (pH{sub 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{sub 4}Cl (acidosis; A) or NaHCO{sub 3} (alkalosis; B) or nothing (control; C) 3 h before the exercise. Muscle pH{sub i} and phosphocreatine (PCr) content were measured with {sup 31}P-nuclear magnetic resonance ({sup 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{sub 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.

  16. The Weak Acid Preservative Sorbic Acid Inhibits Conidial Germination and Mycelial Growth of Aspergillus niger through Intracellular Acidification

    PubMed Central

    Plumridge, Andrew; Hesse, Stephan J. A.; Watson, Adrian J.; Lowe, Kenneth C.; Stratford, Malcolm; Archer, David B.

    2004-01-01

    The growth of the filamentous fungus Aspergillus niger, a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid (trans-trans-2,4-hexadienoic acid). Conidia inoculated at 105/ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pHcyt) by more than 1 pH unit and a depression of vacuolar pH (pHvac) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pHcyt. NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth. PMID:15184150

  17. The sensitivity of a malignant cell line to hyperthermia (42 degrees C) at low intracellular pH.

    PubMed

    Dickson, J A; Oswald, B E

    1976-09-01

    The postulate that low intracellular pH acts as a preconditioner for the destructuve effects of hyperthermia (42 degrees C) was examined, using a heat-sensitive line of malignant cells derived from rat mammary gland (SDB). Intracellular pH (pHi) was measured indirectly, from the distribution of the weak, non-metabolizable organic acid 5,5-dimethyl-2,4-oxazolidinedione (DMO) between intra- and extra-cellular water. Respiration, aerobic and anaerobic and anaerobic glycolysis of the cells were studied at normal pHi (pH 7-0-7-4) or at low pHi (pH 6-2-6-6) and at 38 degrees C or 42 degrees C over 6 h in Warburg manometers; the ability of the cells to replicate in culture was examined after 3 h or 6 h incubation in the flasks. The relationship between pHi and extracellular pH (pHe) depended upon the buffer system used and the exact pH in question; no assumption regarding pHi based only on pHe measurement could be made. At 38 degrees C and low pHi, the Pasteur effect became negative due to a relatively greater inhibition of anaerobic than aerobic glycolysis. Respiration was unaffected and cell replicative ability unimpaired. At 42 degrees C and normal pHi, respiration was totally inhibited after 4 h and the Pasteur effect was decreased, in this case due to a compensatory increase in aerobic glycolysis without alteration in anaerobic CO2 production. Low pHi in the presence of hyperthermia enabled cell respiration to continue at a reduced level with no further change in glycolysis. There was delayed cell replication after 3 h at 42 degrees C and inability to multiply following 6 h hyperthermia: low pHi did not influence these results. It is concluded that with these cancer cells, pHi values maintained in the region of 1-0 pH unit below normal for 6 h had no deleterious effect on the cells. No sensitizing effect of the low pHi for the destructive effect of hyperthermia on the cells was observed. PMID:9969

  18. Control of intracellular pH and growth by fibronectin in capillary endothelial cells

    NASA Technical Reports Server (NTRS)

    Ingber, D. E.; Prusty, D.; Frangioni, J. V.; Cragoe, E. J. Jr; Lechene, C.; Schwartz, M. A.

    1990-01-01

    The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.

  19. Voltage-dependent clamp of intracellular pH of identified leech glial cells.

    PubMed Central

    Deitmer, J W; Schneider, H P

    1995-01-01

    1. The intracellular pH (pHi) was measured in voltage-clamped, giant neuropile glial cells in isolated segmental ganglia of the leech Hirudo medicinalis, using double-barrelled, pH-sensitive microelectrodes and a slow, two-electrode voltage-clamp system. The potential sensitivity of the pHi regulation in these glial cells was found to be due to an electrogenic Na(+)-HCO3- cotransporter (Deitmer & Szatkowski, 1990). 2. In the presence of 5% CO2 and 24 mM HCO3- (pH 7.4), pHi shifted by 1 pH unit per 110 mV, corresponding to a stoichiometry of 2HCO3-: 1 Na+ of the cotransporter, while in Hepes-buffered CO2-HCO3(-)-free saline (pH 7.4), pHi changed by 1 pH unit per 274 mV. The potential sensitivity of pHi decreased at lower pHo, being 1 pH unit per 216 mV at external pH (pHo) 7.0. 3. Changing pHo between 7.8 and 6.6 induced pHi shifts with a slope of 0.72 pHi units per pHo unit in non-clamped, and of 0.80 pHi units per pHo unit in voltage-clamped cells, indicating that pHi largely followed pHo. The electrochemical gradient of H(+)-HCO3- across the glial membrane was around 56 mV, and remained almost constant over this pHo range. 4. The membrane potential-dependent and pHo-sensitive shifts of pHi were unaffected by amiloride, an inhibitor of Na(+)-H+ exchange. 5. The intracellular acidification upon lowering pHo could be reversed by depolarizing the membrane as predicted from a cotransporter, whose equilibrium follows the membrane potential by resetting pHi. 6. The results indicate that the pHi of leech glial cells is dominated by the electrogenic Na(+)-HCO3- cotransporter, and is hence a function of the membrane potential, and the Na+ and H(+)-HCO3- gradients, across the cell membrane. PMID:7658370

  20. Fatty Acid Signaling: The New Function of Intracellular Lipases

    PubMed Central

    Papackova, Zuzana; Cahova, Monika

    2015-01-01

    Until recently, intracellular triacylglycerols (TAG) stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed. PMID:25674855

  1. Hydroxylated near-infrared BODIPY fluorophores as intracellular pH sensors

    PubMed Central

    Salim, Mohamed M.; Owens, Eric A.; Gao, Tielong; Lee, Jeong Heon; Hyun, Hoon; Choi, Hak Soo; Henary, Maged

    2015-01-01

    In this study, a series of new, highly sensitive BF2-chelated tetraarylazadipyrromethane dyes are synthesized and analyzed to be suitable as on/off photo-induced electron transfer modulated fluorescent sensors for determination of intracellular pH. The ethanolic solutions of the new indicators feature absorption maxima in the range of 696–700 nm and a fluorescence emission maximum at 720 nm. Molar absorptivity and fluorescence quantum yield data were determined for the studied set of aza-BODIPY indicators. These indicators have high molar absorption coefficients of ~80 000 M−1 cm−1 and quantum yields (up to 18%). Corresponding pKa values of indicators are determined from absorbance and fluorescence measurements and range from 9.1 to 10.8, depending on the selective positioning of electron-donating functionalities. The excellent photostability of the aza-BODIPY indicators makes them particularly suitable for long duration measurements. The in vitro cellular staining of living tissues in PC3 cells based on the isosbestic point at pH 7.8 and pH 9.3 has been employed which shows an increase in fluorescence intensity at 800 nm with increase in pH for certain compounds and fluorescence intensity decreases at 700 nm. Therefore, the new indicators are suitable for exploitation and adaptation in a diverse range of analytical applications. PMID:25105177

  2. Intracellular Temperature Sensing: An Ultra-bright Luminescent Nanothermometer with Non-sensitivity to pH and Ionic Strength

    PubMed Central

    Liu, Helin; Fan, Yanyan; Wang, Jianhai; Song, Zhongsen; Shi, Hao; Han, Rongcheng; Sha, Yinlin; Jiang, Yuqiang

    2015-01-01

    Luminescence thermometry usually suffer from cellular complexity of the biochemical environment (such as pH and ionic strength), and thus the accuracy and reliability of the determined intracellular temperature are directly affected. Herein, a photoluminescent nanothermometer composed of polymer encapsulated quantum dots (P-QD) has been developed. And the prepared nanothermometer exhibits some advantages: such as non-sensitivity to pH and ionic strength, as well as high detection sensitivity and ultrahigh reversibility. The intracellular temperature was accurately determined under physiological conditions with different pH and ionic strength, and direct measurement of thermogenesis in individual cells has been achieved. PMID:26445905

  3. Intracellular release of doxorubicin from core-crosslinked polypeptide micelles triggered by both pH and reduction conditions.

    PubMed

    Wu, Liangliang; Zou, Yan; Deng, Chao; Cheng, Ru; Meng, Fenghua; Zhong, Zhiyuan

    2013-07-01

    Reduction and pH dual-sensitive reversibly core-crosslinked polypeptide micelles were developed from lipoic acid (LA) and cis-1,2-cyclohexanedicarboxylic acid (CCA) decorated poly(ethylene glycol)-b-poly(L-lysine) (PEG-P(LL-CCA/LA)) block copolymers for active loading and triggered intracellular release of doxorubicin (DOX). PEG-P(LL18-CCA4/LA14) and PEG-P(LL18-CCA8/LA10) (M(n PEG) = 5.0 kg/mol) formed nano-sized micelles that were readily crosslinked in the presence of a catalytic amount of dithiothreitol (DTT) in phosphate buffer (pH 7.4, 10 mM). PEG-P(LL18-CCA4/LA14) micelles displayed an elevated DOX loading over PEG-P(LL14-LA14) controls likely due to presence of ionic interactions between DOX and CCA. These core-crosslinked polypeptide micelles while exhibiting high stability against extensive dilution and high salt concentration were quickly dissociated into unimers in the presence of 10 mM DTT. The in vitro release studies showed that DOX release from PEG-P(LL18-CCA4/LA14) micelles at pH 7.4 and 37 °C was significantly inhibited by crosslinking (i.e. less than 20% release in 24 h). The release of DOX was, however, doubled under endosomal pH of 5.0, possibly triggered by cleavage of the acid-labile amide bonds of CCA. In particular, rapid DOX release was observed under a reductive condition containing 10 mm glutathione (GSH), in which 86.0% and 96.7% of DOX were released in 24 h at pH 7.4 and 5.0, respectively, under otherwise the same conditions. MTT assays demonstrated that these core-crosslinked polypeptide micelles were practically non-toxic up to a tested concentration of 1.0 mg/mL, while DOX-loaded micelles caused pronounced cytotoxic effects to HeLa and HepG2 tumor cells with IC50 (inhibitory concentration to produce 50% cell death) of ca. 12.5 μg DOX equiv/mL following 48 h incubation. Confocal microscopy observations revealed that DOX-loaded crosslinked PEG-P(LL18-CCA4/LA14) micelles more efficiently delivered and released DOX into the nuclei of

  4. Microelectrode study of intracellular pH in frog skin: dependence on serosal Cl/sup -/

    SciTech Connect

    Duffey, M.E.; Kelepouris, E.; Peterson-Yantorno, K.; Civan, M.M.

    1986-03-01

    Replacing external Cl/sup -/ reduces Na/sup +/ transport across frog skin, but the sidedness and mechanisms have been unclear. We have monitored current (l/sub T/), resistance (R/sub T/) and basolateral membrane potential, both with reference micropipettes (psi/sup sc/) and pH-selective microelectrodes (E/sub H//sup sc/), in short-circuited epithelial sheets isolated from frog skins; removal of the dermis facilitates basolateral exchange. Intracellular pH was 7.25 +- 0.03 (mean +- SE) when the pH of the external Cl/sup -/ Ringer's solution was 7.60 +- 0.01, in reasonable agreement with estimates from /sup 31/P and /sup 19/F NMR analyses. Complete mucosal replacement of Cl/sup -/ by gluconate had variable effects on l/sub T/ and R/sub T/. However, serosal Cl/sup -/ substitution uniformly increased R/sub T/ and markedly decreased l/sub T/, absolute value phi/sup sc/ and absolute value E/sub A//sup sc/. The membrane depolarization was usually preceded by a small hyperpolarization (0.5-3.5 mV). The serosal Cl/sup -/ replacement also produced an intracellular alkalinization of 0.4 +- 0.1 U. These data suggest that: (1) serosal Cl/sup -/ substitution alkalinizes the cells by either enhancing HCO/sup -/ entry or blocking HCO/sup -/ loss through a basolateral Cl/HCO antiport, and (2) the fall in absolute value phi/sup sc/ and l/sub T/ may partly reflect inhibition of apical Na/sup +/ entry, produced indirectly by membrane depolarization resulting from altered basolateral ionic conductances.

  5. Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors.

    PubMed

    Boron, W F; De Weer, P

    1976-01-01

    The intracellular pH (pHi) of squid giant axons has been measured using glass pH microelectrodes. Resting pHi in artificial seawater (ASW) (pH 7.6-7.8) at 23 degrees C was 7.32 +/- 0.02 (7.28 if corrected for liquid junction potential). Exposure of the axon to 5% CO2 at constant external pH caused a sharp decrease in pHi, while the subsequent removal of the gas caused pHi to overshoot its initial value. If the exposure to CO2 was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial fall in pHi was followed by a slow rise, and (b) after the exposure, the overshoot was greatly exaggerated. Application of external NH4Cl caused pHi to rise sharply; return to normal ASW caused pHi to return to a value below its initial one. If the exposure to NH4Cl was prolonged, two additional effects were noted: (a) during the exposure, the rapid initial rise in pHi was followed by a slow fall, and (b) after the exposure, the undershoot was greatly exaggerated. Exposure to several weak acid metabolic inhibitors caused a fall in pHi whose reversibility depended upon length of exposure. Inverting the electrochemical gradient for H+ with 100 mM K-ASW had no effect on pHi changes resulting from short-term exposure to azide. A mathematical model explains the pHi changes caused by NH4Cl on the basis of passive movements of both NH3 and NH4+. The simultaneous passive movements of CO2 and HCO3-cannot explain the results of the CO2 experiments; these data require the postulation of an active proton extrusion and/or sequestration mechanism. PMID:1460

  6. Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton

    PubMed Central

    LI, JIEJING; LI, YANXI; ZHANG, PENGHUI; NIU, HUA; SHI, YU

    2014-01-01

    Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F-actin cytoskeleton systems and resulted in β-tubulin degradation in an ubiquitin-proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F-actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F-actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport. PMID:25241762

  7. Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton.

    PubMed

    Li, Jiejing; Li, Yanxi; Zhang, Penghui; Niu, Hua; Shi, Yu

    2014-12-01

    Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F‑actin cytoskeleton systems and resulted in β‑tubulin degradation in an ubiquitin‑proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F‑actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F‑actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport. PMID:25241762

  8. pH [Measure of Acidity].

    ERIC Educational Resources Information Center

    Henderson, Paula

    This autoinstructional program deals with the study of the pH of given substances by using litmus and hydrion papers. It is a learning activity directed toward low achievers involved in the study of biology at the secondary school level. The time suggested for the unit is 25-30 minutes (plus additional time for further pH testing). The equipment…

  9. 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.

    PubMed

    Orij, Rick; Postmus, Jarne; Ter Beek, Alex; Brul, Stanley; Smits, Gertien 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 understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH (pHcyt) and mitochondrial pH (pHmit) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant pHcyt of 7.2 and a constant pHmit of 7.5 in cells exponentially growing on glucose. We show that pHcyt and pHmit are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, pHi decrease coincided with growth stasis. Additionally, pHi and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pHi and cellular energy generation, and therefore a relation between pHi and growth. PMID:19118367

  10. Effect of potassium depletion of Hep 2 cells on intracellular pH and on chloride uptake by anion antiport

    SciTech Connect

    Madshus, I.H.; Tonnessen, T.I.; Olsnes, S.; Sandvig, K.

    1987-04-01

    The effect of K+ depletion of Hep 2 cells on ion fluxes, internal pH, cell volume, and membrane potential was studied. The cells were depleted of K+ by incubation in K+-free buffer with or without a preceding exposure to hypotonic medium. Efflux of K+ in cells not exposed to hypotonic medium was inhibited by furosemide or by incubation in Na+-free medium, indicating that in this case at least part of the K+ efflux occurs by Na+/K+/Cl- cotransport. After exposure to hypotonic medium, K+ efflux was not inhibited by furosemide, whereas it was partly inhibited by 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS). Exposure to hypotonic medium induced acidification of the cytosol, apparently because of efflux of protons from intracellular acidic vesicles. When isotonicity was restored, a rebound alkalinization of the cytosol was induced, because of activation of the Na+/H+ antiporter. While hypotonic shock and a subsequent incubation in K+-free buffer rapidly depolarized the cells, depolarization occurred much more slowly when the K+ depletion was carried out by incubation in K+-free buffer alone. The cell volume was reduced in both cases. K+ depletion by either method strongly reduced the ability of the cells to accumulate /sup 36/Cl- by anion antiport, and K+-depleted cells were unable to increase the rate of /sup 36/Cl- uptake in response to alkalinization of the cytosol.

  11. Cytotoxic effect of RB 6145 in human tumour cell lines: dependence on hypoxia, extra- and intracellular pH and drug uptake.

    PubMed Central

    Skarsgard, L. D.; Acheson, D. K.; Vinczan, A.; Wouters, B. G.; Heinrichs, B. E.; Loblaw, D. A.; Minchinton, A. I.; Chaplin, D. J.

    1995-01-01

    Low pH and hypoxia are a common feature of many solid tumours. This study examined the effect of these two conditions on the cytotoxic properties of the bifunctional agent RB 6145, the prodrug of RSU 1069. The effect of acidic pH on RB 6145 toxicity was examined in six human tumour cell lines under hypoxic conditions and was found to have little effect in HT 29, A549, U373 and HT 144 cells. Treatment was for 1 h at 37 degrees C, pH 6.4 or 7.4. Significant potentiation of RB 6145 toxicity was observed in SiHa cells (enhancement ratio; ERpH approximately 1.6) and in U1 cells (ERpH approximately 1.4). In these two cell lines the potentiation of RB 6145 toxicity arising from hypoxia was large, with ERHyp approximately 11 and 15 in SiHa and U1 cells respectively. SiHa cells, which show a pH effect and HT 29 cells, which do not, were chosen for further comparative studies of drug uptake )nd regulation of intracellular pH. High-performance liquid chromatography (HPLC) determinations of the uptake of RB 6145 and its dervatives showed that in SiHa cells, intracellular to extracellular drug concentration ratio (Ci/Ce) at 1 h was approximately 40% higher at pH 6.4 than at pH 7.4, whereas in HT 29 cells Ci/Ce was approximately 25% lower. Under conditions of acidic extracellular pH, regulation of pH was somewhat less effective in SiHa cells, where pHi dropped to within 0.2 pH units of the extracellular pH over a 2.5 h treatment at pH 6.4. It seems likely that increased drug uptake was at least part of the basis for the observed potentiation of RB 6145 toxicity in SiHa cells. A model which would better explain the results for both cell lines might also include the possibility that low pH per se potentiates cytotoxic damage to a modest extent and that it is offset or augmented by altered uptake in HT 29 and SiHa cells respectively. PMID:8519663

  12. Glucose Uptake and Intracellular pH in a Mouse Model of Ductal Carcinoma In situ (DCIS) Suggests Metabolic Heterogeneity

    PubMed Central

    Lobo, Rebecca C.; Hubbard, Neil E.; Damonte, Patrizia; Mori, Hidetoshi; Pénzváltó, Zsófia; Pham, Cindy; Koehne, Amanda L.; Go, Aiza C.; Anderson, Steve E.; Cala, Peter M.; Borowsky, Alexander D.

    2016-01-01

    Mechanisms for the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinoma remain unclear. Previously we showed that the transition to invasiveness in the mammary intraepithelial neoplastic outgrowth (MINO) model of DCIS does not correlate with its serial acquisition of genetic mutations. We hypothesized instead that progression to invasiveness depends on a change in the microenvironment and that precancer cells might create a more tumor-permissive microenvironment secondary to changes in glucose uptake and metabolism. Immunostaining for glucose transporter 1 (GLUT1) and the hypoxia marker carbonic anhydrase 9 (CAIX) in tumor, normal mammary gland and MINO (precancer) tissue showed differences in expression. The uptake of the fluorescent glucose analog dye, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), reflected differences in the cellular distributions of glucose uptake in normal mammary epithelial cells (nMEC), MINO, and Met1 cancer cells, with a broad distribution in the MINO population. The intracellular pH (pHi) measured using the fluorescent ratio dye 2′,7′-bis(2-carboxyethyl)-5(6)-155 carboxyfluorescein (BCECF) revealed expected differences between normal and cancer cells (low and high, respectively), and a mixed distribution in the MINO cells, with a subset of cells in the MINO having an increased rate of acidification when proton efflux was inhibited. Invasive tumor cells had a more alkaline baseline pHi with high rates of proton production coupled with higher rates of proton export, compared with nMEC. MINO cells displayed considerable variation in baseline pHi that separated into two distinct populations: MINO high and MINO low. MINO high had a noticeably higher mean acidification rate compared with nMEC, but relatively high baseline pHi similar to tumor cells. MINO low cells also had an increased acidification rate compared with nMEC, but with a more acidic pHi similar to nMEC. These findings

  13. Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress

    NASA Technical Reports Server (NTRS)

    Stamatas, G. N.; Patrick, C. W. Jr; McIntire, L. V.

    1997-01-01

    The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.

  14. Axial heterogeneity of intracellular pH in rat proximal convoluted tubule.

    PubMed Central

    Pastoriza-Munoz, E; Harrington, R M; Graber, M L

    1987-01-01

    In the proximal convoluted tubule (PT), the HCO3- reabsorptive rate is higher in early (EPS) compared with late proximal segments (LPS). To examine the mechanism of this HCO3- reabsorption profile, intracellular pH (pHi) was measured along the superficial PT of the rat under free-flow and stationary microperfusion using the pH-sensitive fluorescence of 4-methylumbelliferone (4MU). With 4MU superfusion, pHi was found to decline along the PT. Observation with 365-nm excitation revealed that EPS were brightly fluorescent and always emerged away from their star vessel. Midproximal segments were darker and closer to the star vessel which was surrounded by the darkest LPS. Decreasing luminal HCO3- from 15 to 0 mM lowered pHi in both EPS and LPS, but pHi remained more alkaline in EPS with both perfusates. Thus the axial decline in pHi along the PT is due to both luminal factors and intrinsic differences in luminal H+ extrusion in PT cells. PMID:3036912

  15. Fluorescent probes in biology and medicine: measurement of intracellular pH values in individual cells

    NASA Astrophysics Data System (ADS)

    Slavik, Jan; Cimprich, Petr; Gregor, Martin; Smetana, Karel, Jr.

    1997-12-01

    The application possibilities of fluorescent probes have increased dramatically in the last few years. The main areas are as follows (Slavik, 1994, 1996, 1998). Intracellular ionic cell composition: There are selective ion-sensitive dyes for H+, Ca2+, Mg2+, K+, Na+, Fe3+, Cl-, Zn2+, Cd2+, Hg2+, Pb2+, Ba2+, La3+. Membrane potential: Using the so-called slow (Nernstian dyes) or electrochromic dyes one can assess the value of the transmembrane potential. Membrane fluidity: Fluorescent probes inform about the freedom of rotational and translational movement of membrane proteins and lipids. Selective labeling: Almost any object of interest inside the cell or on its surface can be selectively fluorescently labeled. There are dyes specific for DNA, RNA, oligonucleotides (FISH), Golgi, endoplasmic reticulum, mitochondria, vacuoles, cytoskeleton, etc. Using fluorescent dyes specific receptors may be localized, their conformational changes followed and the polarity of corresponding binding sites accessed. The endocytic pathway may be followed, enzymes and their local enzymatic activity localized. For really selective labeling fluorescent labeled antibodies exist. Imaging: One of the main advantages of fluorescence imaging is its versatility. It allow choice among ratio imaging in excitation, ratio imaging in emission and lifetime imaging. These approaches can be applied to both the classical wide-field fluorescence microscopy and to the laser confocal fluorescence microscopy, one day possibly to the scanning near field optical microscopy. Simultaneous application of several fluorescent dyes: The technical progress in both excitation sources and in detectors allows to extend the excitation deeper in the blue and ultraviolet side and the detection further in the NIR and IR. Consequently, up to 6 peaks in excitation and up to 6 peaks in emission can be followed without any substantial difficulties. Application of dyes such with longer fluorescence lifetimes such as rare earth

  16. 31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

    PubMed

    Civan, M M; Williams, S R; Gadian, D G; Rozengurt, E

    1986-01-01

    Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event. PMID:3543375

  17. Gold nanoparticles with different amino acid surfaces: serum albumin adsorption, intracellular uptake and cytotoxicity.

    PubMed

    Cai, Huanxin; Yao, Ping

    2014-11-01

    Gold nanoparticles with aspartate, glycine, leucine, lysine, and serine surfaces were produced from the mixed solutions of HAuCl4 and respective amino acids via UV irradiation. The amino acids bind to the nanoparticle surfaces via amine groups and their carboxylic groups extend out to stabilize the nanoparticles. The nanoparticles have diameters of 15-47 nm in pH 7.4 aqueous solution and have diameters of 62-73 nm after 48 h incubation in cell culture containing serum. The nanoparticles adsorb human and bovine serum albumins on their surfaces by specific interactions, characterized by the intrinsic fluorescence quenching of the albumins. The albumin adsorption effectively decreases the aggregation of the nanoparticles in cell culture and also decreases the intracellular uptake of the nanoparticles. The gold nanoparticles produced from leucine and lysine, which have amphiphilic groups on their surfaces, present better biocompatibility than the other gold nanoparticles. PMID:25466455

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

    PubMed Central

    Benčina, Mojca

    2013-01-01

    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. PMID:24316570

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

    PubMed

    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. PMID:27424899

  20. Muscle fatigue in frog semitendinosus: role of intracellular pH.

    PubMed

    Thompson, L V; Balog, E M; Fitts, R H

    1992-06-01

    The purpose of this study was to utilize glass microelectrodes to characterize the intracellular pH (pHi) before and during recovery from fatigue in the frog semitendinosus (ST) muscle. A second objective was to evaluate the relationship between pHi and contractile function. The frog ST muscle (22 degrees C) was fatigued by direct electrical stimulation with 100-ms 150-Hz trains at 1/s for 5 min. Peak tetanic force (Po) was reduced to 8.5% of initial force and recovered in a biphasic manner, returning to the resting value by 40 min. Resting pHi was 7.00 +/- 0.02 (n = 37) and declined with fatigue to an average value of 6.42 at 3 min of recovery. During recovery pHi significantly increased and by 25 min had returned to the prefatigue value. The pHi recovery was highly correlated to the slow phase of Po recovery (r = 0.98, P less than 0.001). The mean resting membrane potential was -78 +/- 1.0 mV (n = 42) and at 3 min of recovery was depolarized to -67 +/- 4 mV. Both the peak rate of twitch force development (+dP/dt) (r = 0.99, P less than 0.001) and decline (-dP/dt) (r = 0.94, P less than 0.014) were highly correlated to pHi during the slow phase of recovery. Contraction time (CT) and one-half relaxation time (1/2RT) increased significantly and recovered exponentially. The recovery of CT and 1/2RT were both significantly correlated to pHi (r = -0.93, P less than 0.001 and r = -0.86, P less than 0.001 for CT and 1/2RT, respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1616012

  1. Muscle fatigue in frog semitendinosus: role of intracellular pH

    NASA Technical Reports Server (NTRS)

    Thompson, L. V.; Balog, E. M.; Fitts, R. H.

    1992-01-01

    The purpose of this study was to utilize glass microelectrodes to characterize the intracellular pH (pHi) before and during recovery from fatigue in the frog semitendinosus (ST) muscle. A second objective was to evaluate the relationship between pHi and contractile function. The frog ST muscle (22 degrees C) was fatigued by direct electrical stimulation with 100-ms 150-Hz trains at 1/s for 5 min. Peak tetanic force (Po) was reduced to 8.5% of initial force and recovered in a biphasic manner, returning to the resting value by 40 min. Resting pHi was 7.00 +/- 0.02 (n = 37) and declined with fatigue to an average value of 6.42 at 3 min of recovery. During recovery pHi significantly increased and by 25 min had returned to the prefatigue value. The pHi recovery was highly correlated to the slow phase of Po recovery (r = 0.98, P less than 0.001). The mean resting membrane potential was -78 +/- 1.0 mV (n = 42) and at 3 min of recovery was depolarized to -67 +/- 4 mV. Both the peak rate of twitch force development (+dP/dt) (r = 0.99, P less than 0.001) and decline (-dP/dt) (r = 0.94, P less than 0.014) were highly correlated to pHi during the slow phase of recovery. Contraction time (CT) and one-half relaxation time (1/2RT) increased significantly and recovered exponentially. The recovery of CT and 1/2RT were both significantly correlated to pHi (r = -0.93, P less than 0.001 and r = -0.86, P less than 0.001 for CT and 1/2RT, respectively).(ABSTRACT TRUNCATED AT 250 WORDS).

  2. Proliferation and intracellular pH in cultured proximal tubular cells

    SciTech Connect

    Larsson, S.H.; Fukuda, Y.; Koelare, S.A.; Aperia, A. )

    1990-03-01

    Renal proximal tubule (PT) cells from adult rats will maintain much of their functional characteristics in short-term primary culture. This study examines the growth regulation of these highly differentiated cells with particular reference to cell density, intracellular pH (pHi), and the expression of the Na(+)-H+ exchanger. PT cells were obtained from young adult rats and studied after 48 h in culture. The mitotic rate was determined as the labeling index (LI) after (3H)thymidine autoradiography, and pHi was determined by 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein quantitative fluorescence microscopy in single cells. Cells were grown either continuously in serum (S) or were serum deprived after 24 h (D). The cells were nonconfluent and grew in colonies. We defined the two peripheral layers of cells in a colony as peripheral (P) cells and the remaining cells as central (C). In C cells LI/h and pHi were in the range of what has been observed under in vivo conditions. In S condition LI/h was 2.2 +/- 0.3% and in D condition was 0.3 +/- 0.1%. LI was significantly higher in P than in C cells both under S (2.5 +/- 0.4-fold) and D conditions (5.6 +/- 0.8-fold). The rapidly growing P cells had a significantly lower pHi than the growth-retarded C cells both under S (7.25 +/- 0.02 vs. 7.30 +/- 0.01, P less than 0.05) and D conditions (7.21 +/- 0.02 vs. 7.28 +/- 0.01, P less than 0.05).

  3. Thermally induced changes in intracellular pH and modulators of phosphofructokinase in trout white muscle

    PubMed

    Lehoux; Guderley

    1997-01-01

    The intracellular pH (pHi) and the concentrations of lactate and selected modulators of phosphofructokinase (PFK; EC 2.7.1.11) were measured in white epaxial muscle of 15 °C-acclimated rainbow trout (Oncorhynchus mykiss) maintained at 8, 15 or 22 °C for 48 h and sampled at rest and after 10 min of exhaustive exercise. The lactate accumulation resulting from exercise was 13 % smaller at 22 °C than at 8 and 15 °C. The estimated duration of burst performance was shorter at cold than at warm temperatures, whereas the average rate of lactate accumulation during burst performance was higher at 8 °C than at 15 and 22 °C. pHi rose when temperature decreased, but less than predicted by the imidazole alphastat hypothesis of Reeves. The effects of temperature on the pre-exercise concentrations of PFK modulators [adenylates, fructose 6-phosphate (F6P) and fructose 1,6-bisphosphate (FBP)] were generally negligible. In exhausted trout, adenylate concentrations were almost unaffected by temperature. In contrast, post-exercise FBP and F6P concentrations were significantly higher at low than at high temperatures. We interpret the response of F6P to temperature as an indication that the covariation of pHi and temperature is insufficient to prevent a cold-enhancement of PFK inhibition. Since F6P is a potent activator of PFK, we conclude that, in trout white muscle, thermally induced changes in F6P concentration probably help buffer the effects of temperature change on PFK activity. PMID:9318724

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

    PubMed Central

    Bröer, Angelika; Albers, Alexandra; Setiawan, Iwan; Edwards, Robert H; Chaudhry, Farrukh A; Lang, Florian; Wagner, Carsten A; Bröer, Stefan

    2002-01-01

    The glutamine transporter 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 system N amino acid transport. Two different transport mechanisms have been proposed for this transporter. These are 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 these discrepancies and to investigate the reversibility of the transporter. When SN1 was expressed in Xenopus laevis oocytes, glutamine uptake was accompanied by a cotransport of 2–3 Na+ ions as determined by 22Na+ fluxes. However, at the same time a rapid release of intracellular Na+ was observed indicating an active exchange of Na+ ions. The driving force of the proton electrochemical gradient was equivalent to that of the sodium electrochemical gradient. Acidification of the extracellular medium caused the transporter to run in reverse and to release glutamine. Determination of accumulation ratios at different driving forces were in agreement with an electroneutral 1Na+-glutamine cotransport-1H+ antiport. Inward currents that were observed during glutamine uptake were much smaller than expected for a stoichiometric cotransport of charges. A slippage mode in the transporter mechanism and pH-regulated endogenous oocyte cation channels are likely to contribute to the observed currents. PMID:11850497

  5. Effects of Fatty Acids on Intracellular [Ca2+], Mitochondrial Uncoupling and Apoptosis in Rat Pachytene Spermatocytes and Round Spermatids

    PubMed Central

    Paillamanque, Joaquín; Madrid, Cristian; Carmona, Emerson M.; Osses, Nelson; Moreno, Ricardo D.; Oresti, Gerardo M.; Pino, José A.

    2016-01-01

    The aim of this work was to explore the ability of free arachidonic acid, palmitic acid and the unsaturated fatty acids oleic acid and docosahexaenoic acid to modify calcium homeostasis and mitochondrial function in rat pachytene spermatocytes and round spermatids. In contrast to palmitic acid, unsaturated fatty acids produced significant increases in intracellular calcium concentrations ([Ca2+]i) in both cell types. Increases were fatty acid specific, dose-dependent and different for each cell type. The arachidonic acid effects on [Ca2+]i were higher in spermatids than in spermatocytes and persisted when residual extracellular Ca2+ was chelated by EGTA, indicating that the increase in [Ca2+]i originated from release of intracellular calcium stores. At the concentrations required for these increases, unsaturated fatty acids produced no significant changes in the plasma membrane potential of or non-specific permeability in spermatogenic cells. For the case of arachidonic acid, the [Ca2+]i increases were not caused by its metabolic conversion to eicosanoids or anandamide; thus we attribute this effect to the fatty acid itself. As estimated with fluorescent probes, unsaturated fatty acids did not affect the intracellular pH but were able to induce a progressive decrease in the mitochondrial membrane potential. The association of this decrease with reduced reactive oxygen species (ROS) production strongly suggests that unsaturated fatty acids induced mitochondrial uncoupling. This effect was stronger in spermatids than in spermatocytes. As a late event, arachidonic acid induced caspase 3 activation in a dose-dependent manner both in the absence and presence of external Ca2+. The concurrent but differential effects of unsaturated fatty acids on [Ca2+]i and mitochondrial functions are additional manifestations of the metabolic changes that germ cells undergo during their differentiation. PMID:27428262

  6. CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm.

    PubMed

    Loux, Shavahn C; Crawford, Kristin R; Ing, Nancy H; González-Fernández, Lauro; Macías-García, Beatriz; Love, Charles C; Varner, Dickson D; Velez, Isabel C; Choi, Young Ho; Hinrichs, Katrin

    2013-11-01

    In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. In calcium-deficient medium, high-pH treatment induced motility loss, consistent with influx of sodium through open CatSper channels in the absence of environmental calcium. However, sperm treated with procaine in calcium-deficient medium both maintained motility and underwent hyperactivation, suggesting that procaine did not act via opening of the CatSper channel. CATSPER1 mRNA was identified in equine sperm by PCR, and CATSPER1 protein was localized to the principal piece on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region. We conclude that the CatSper channel is present in equine sperm but that the relationship of hyperactivated motility to calcium influx is weak. Procaine does not appear to act via CatSper in equine sperm, and its initial hyperactivating action is not dependent upon external calcium influx. PMID:24048572

  7. Novel pH-sensitive polysialic acid based polymeric micelles for triggered intracellular release of hydrophobic drug.

    PubMed

    Zhang, Wuxia; Dong, Dongqi; Li, Peng; Wang, Dongdong; Mu, Haibo; Niu, Hong; Duan, Jinyou

    2016-03-30

    Polysialic acid (PSA), a non-immunogenic and biodegradable natural polymer, is prone to hydrolysis under endo-lysosomal pH conditions. Here, we synthesized an intracellular pH-sensitive polysialic acid-ursolic acid conjugate by a condensation reaction. To further test the drug loading capability, we prepared paclitaxel-loaded polysialic acid-based amphiphilic copolymer micelle (PTX-loaded-PSAU) by a nanoprecipitation method. Results showed PTX-loaded-PSAU exhibited well-defined spherical shape and homogeneous distribution. The drug-loading was 4.5% with an entrapment efficiency of 67.5%. PTX released from PTX-loaded-PSAU was 15% and 42% in 72 h under simulated physiological condition (pH 7.4) and mild acidic conditions (pH 5.0), respectively. In addition, In vitro cytotoxicity assay showed that PTX-loaded-PSAU retained anti-tumor (SGC-7901) activity with a cell viability of 53.8% following 72 h incubation, indicating PTX-loaded-PSAU could efficiently release PTX into the tumor cells. These results indicated that the pH-responsive biodegradable PTX-loaded-PSAU possess superior extracellular stability and intracellular drug release ability. PMID:26794949

  8. [Ultrasonic study of nucleic acids. Effect of pH].

    PubMed

    Braginskaia, F I; Sadykhova, S Kh

    1979-01-01

    The ultrasonic absorption of nucleic acids in water solutions was studied by the pulse ultrasonic technique depending on pH, at frequency 12 mHz T = 20 dedrees C. The obtained data show the occurrence of structural relaxation in DNA solutions caused by the proton exchange and transfer reactions with the extremal pH at 2.5 and 11.7. Possible mechanisms of the excess ultrasonic absorption were discussed concerning the protolytic processes with the charged DNA groups (N--P1 exchange and the hydrolysis of lactam groups at acid and alkaline pH correspondingly). PMID:36177

  9. Parasitophorous vacuoles of Leishmania amazonensis-infected macrophages maintain an acidic pH.

    PubMed Central

    Antoine, J C; Prina, E; Jouanne, C; Bongrand, P

    1990-01-01

    Leishmania amastigotes are intracellular protozoan parasites of mononuclear phagocytes which reside within parasitophorous vacuoles of phagolysosomal origin. The pH of these compartments was studied with the aim of elucidating strategies used by these microorganisms to evade the microbicidal mechanisms of their host cells. For this purpose, rat bone marrow-derived macrophages were infected with L. amazonensis amastigotes. Intracellular acidic compartments were localized by using the weak base 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine as a probe. This indicator, which can be detected by light microscopy by using immunocytochemical methods, mainly accumulated in perinuclear lysosomes of uninfected cells, whereas in infected cells, it was essentially localized in parasitophorous vacuoles, which thus appeared acidified. Phagolysosomal pH was estimated quantitatively in living cells loaded with the pH-sensitive endocytic tracer fluoresceinated dextran. After a 15- to 20-h exposure, the tracer was mainly detected in perinuclear lysosomes and parasitophorous vacuoles of uninfected and infected macrophages, respectively. Fluorescence intensities were determined from digitized video images of single cells after processing and automatic subtraction of background. We found statistically different mean pH values of 5.17 to 5.48 for lysosomes and 4.74 to 5.26 for parasitophorous vacuoles. As for lysosomes of monensin-treated cells, the pH gradient of parasitophorous vacuoles collapsed after monensin was added. This very likely indicates that these vacuoles maintain an acidic internal pH by an active process. These results show that L. amazonensis amastigotes are acidophilic and opportunistic organisms and suggest that these intracellular parasites have evolved means for survival under these harsh conditions and have acquired plasma membrane components compatible with the environment. Images PMID:1689700

  10. Antithrombotic activities of ferulic acid via intracellular cyclic nucleotide signaling.

    PubMed

    Hong, Qian; Ma, Zeng-Chun; Huang, Hao; Wang, Yu-Guang; Tan, Hong-Ling; Xiao, Cheng-Rong; Liang, Qian-De; Zhang, Han-Ting; Gao, Yue

    2016-04-15

    Ferulic acid (FA) produces protective effects against cardiovascular dysfunctions. However, the mechanisms of FA is still not known. Here we examined the antithrombotic effects of FA and its potential mechanisms. Anticoagulation assays and platelet aggregation was evaluated in vitro and in vivo. Thromboxane B2 (TXB2), cyclic adenosine monophosphate(cAMP), and cyclic guanosine monophosphate (cGMP) was determined using enzyme immunoassay kits. Nitric oxide (NO) production was measured using the Griess reaction. Protein expression was detected by Western blotting analysis. Oral administration of FA prevented death caused by pulmonary thrombosis and prolonged the tail bleeding and clotting time in mice,while, it did not alter the coagulation parameters, including the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). In addition, FA (50-200µM) dose-dependently inhibited platelet aggregation induced by various platelet agonists, including adenosine diphosphate (ADP), thrombin, collagen, arachidonic acid (AA), and U46619. Further, FA attenuated intracellular Ca(2)(+) mobilization and TXB2 production induced by the platelet agonists. FA increased the levels of cAMP and cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP) while decreased phospho-MAPK (mitogen-activated protein kinase) and phosphodiesterase (PDE) in washed rat platelets, VASP is a substrate of cyclic nucleotide and PDE is an enzyme family responsible for hydrolysis of cAMP/cGMP. These results suggest that antithrombotic activities of FA may be regulated by inhibition of platelet aggregation, rather than through inhibiting the release of thromboplastin or formation of thrombin. The mechanism of this action may involve activation of cAMP and cGMP signaling. PMID:26948317

  11. Phosphorus-31 nuclear magnetic resonance study of post mortem catabolism and intracellular pH in intact excised rabbit muscle.

    PubMed

    Renou, J P; Canioni, P; Gatelier, P; Valin, C; Cozzone, P J

    1986-04-01

    Phosphorus-31 nuclear magnetic resonance has been used to study the post mortem catabolism of high-energy phosphate compounds and the associated intracellular pH variation in pure fast- and slow-twitch rabbit muscles and in rabbit muscle with mixed fiber types. Comparative results from pure fiber types are reported for the first time. Large amounts of glycerophosphorylcholine (14.1 mumol/g fresh tissue) are found in the internal conoidal bundle (ICB), a pure oxidative slow twitch muscle, whereas the m. psoas major (PM), a pure glycolytic fast twitch muscle and the m. gastrocnemius caput medialis (GCM), with mixed fiber types, are devoid of the same metabolite. The total content of phosphorylated metabolites is constant among the three muscle types. The time-dependent post mortem changes in phosphorylated metabolites display the expected rapid drop in phosphocreatine and a simultaneous increase in intracellular inorganic phosphate. However, the ATP level remains constant during more than 2 h. Rate constants for metabolite breakdown and apparent ATPase activity have been determined. The comparative kinetics of intracellular acidosis at 25 degrees C yield rates of 3.3 X 10(-3) pH unit/min for PM, 2.7 X 10(-3) pH unit/min for GCM and 3.0 X 10(-3) pH unit/min for ICB. Initial intracellular pH values are 7.07, 7.20 and 7.02, respectively. Upon aging, the heterogeneity of the Pi signal reflects the existence of cellular compartments with different internal pH. The results suggest that the more intense low-pH Pi signal arises from the sarcoplasmic reticulum while the less intense resonance would reflect the sarcoplasmic higher pH. The temperature effect on post mortem catabolism in the 15-25 degrees C range has been documented. As expected, phosphocreatine and ATP breakdown increase with temperature but at a higher rate for slow-twitch ICB than for fast-twitch PM. PMID:3091088

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

    NASA Astrophysics Data System (ADS)

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

    2013-09-01

    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 λex-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 pKa 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.

  13. Titratable acidity of beverages influences salivary pH recovery.

    PubMed

    Tenuta, Livia Maria Andaló; Fernández, Constanza Estefany; Brandão, Ana Carolina Siqueira; Cury, Jaime Aparecido

    2015-01-01

    A low pH and a high titratable acidity of juices and cola-based beverages are relevant factors that contribute to dental erosion, but the relative importance of these properties to maintain salivary pH at demineralizing levels for long periods of time after drinking is unknown. In this crossover study conducted in vivo, orange juice, a cola-based soft drink, and a 10% sucrose solution (negative control) were tested. These drinks differ in terms of their pH (3.5 ± 0.04, 2.5 ± 0.05, and 5.9 ± 0.1, respectively) and titratable acidity (3.17 ± 0.06, 0.57 ± 0.04 and < 0.005 mmols OH- to reach pH 5.5, respectively). Eight volunteers with a normal salivary flow rate and buffering capacity kept 15 mL of each beverage in their mouth for 10 s, expectorated it, and their saliva was collected after 15, 30, 45, 60, 90, and 120 s. The salivary pH, determined using a mini pH electrode, returned to the baseline value at 30 s after expectoration of the cola-based soft drink, but only at 90 s after expectoration of the orange juice. The salivary pH increased to greater than 5.5 at 15 s after expectoration of the cola drink and at 30 s after expectoration of the orange juice. These findings suggest that the titratable acidity of a beverage influences salivary pH values after drinking acidic beverages more than the beverage pH. PMID:25715032

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

    PubMed Central

    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

  15. Croconaine rotaxane for acid activated photothermal heating and ratiometric photoacoustic imaging of acidic pH.

    PubMed

    Guha, Samit; Shaw, Gillian Karen; Mitcham, Trevor M; Bouchard, Richard R; Smith, Bradley D

    2016-01-01

    Absorption of 808 nm laser light by liposomes containing a pH sensitive, near-infrared croconaine rotaxane dye increases dramatically in weak acid. A stealth liposome composition permits acid activated, photothermal heating and also acts as an effective nanoparticle probe for ratiometric photoacoustic imaging of acidic pH in deep sample locations, including a living mouse. PMID:26502996

  16. pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH

    PubMed Central

    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. PMID:22905204

  17. Intracellular fate of spherical nucleic acid nanoparticle conjugates.

    PubMed

    Wu, Xiaochen A; Choi, Chung Hang J; Zhang, Chuan; Hao, Liangliang; Mirkin, Chad A

    2014-05-28

    Spherical nucleic acid (SNA) nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications. Their unique ability to enter multiple mammalian cell types as single-entity agents arises from their novel three-dimensional architecture, which consists of a dense shell of highly oriented oligonucleotides chemically attached typically to a gold nanoparticle core. This architecture allows SNAs to engage certain cell surface receptors to facilitate entry. Here, we report studies aimed at determining the intracellular fate of SNAs and the trafficking events that occur inside C166 mouse endothelial cells after cellular entry. We show that SNAs traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation. Disassembly of oligonucleotides from the nanoparticle core is observed 16 h after cellular entry, most likely due to degradation by enzymes such as DNase II localized in late endosomes. Our observations point to these events being likely independent of core composition and treatment conditions, and they do not seem to be particularly dependent upon oligonucleotide sequence. Significantly and surprisingly, the SNAs do not enter the lysosomes under the conditions studied. To independently track the fate of the particle core and the fluorophore-labeled oligonucleotides that comprise its shell, we synthesized a novel class of quantum dot SNAs to determine that as the SNA structures are broken down over the 24 h time course of the experiment, the oligonucleotide fragments are recycled out of the cell while the nanoparticle core is not. This mechanistic insight points to the importance of designing and synthesizing next-generation SNAs that can bypass the degradation bottleneck imposed by their residency in late endosomes, and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome

  18. Dehydroascorbic acid uptake by coronary artery smooth muscle: effect of intracellular acidification.

    PubMed Central

    Holmes, Melanie E; Mwanjewe, James; Samson, Sue E; Haist, James V; Wilson, John X; Dixon, S Jeffrey; Karmazyn, Morris; Grover, Ashok K

    2002-01-01

    Dehydroascorbic acid (DHAA) enters cells via Na(+)-independent glucose transporters (GLUT) and is converted to ascorbate. However, we found that Na(+) removal inhibited [(14)C]DHAA uptake by smooth-muscle cells cultured from pig coronary artery. The uptake was examined for 2-12 min at 10-200 microM DHAA in either the presence of 134 mM Na(+) or in its absence (N-methyl D-glucamine, choline or sucrose replaced Na(+)). This inhibition of DHAA uptake by Na(+) removal was paradoxical because it was inhibited by 2-deoxyglucose and cytochalasin B, as expected of transport via the GLUT pathway. We tested the hypothesis that this paradox resulted from an inefficient intracellular reduction of [(14)C]DHAA into [(14)C]ascorbate upon intracellular acidosis caused by the Na(+) removal. Consistent with this hypothesis: (i) the Na(+)/H(+)-exchange inhibitors ethylisopropyl amiloride and cariporide also decreased the uptake, (ii) Na(+) removal and Na(+)/H(+)-exchange inhibitors lowered cytosolic pH, with the decrease being larger in 12 min than in 2 min, and (iii) less of the cellular (14)C was present as ascorbate (determined by HPLC) in cells in Na(+)-free buffer than in those in Na(+)-containing buffer. This inability to obtain ascorbate from extracellular DHAA may be detrimental to the coronary artery under hypoxia-induced acidosis during ischaemia/reperfusion. PMID:11853561

  19. Life at acidic pH imposes an increased energetic cost for a eukaryotic acidophile.

    PubMed

    Messerli, Mark A; Amaral-Zettler, Linda A; Zettler, Erik; Jung, Sung-Kwon; Smith, Peter J S; Sogin, Mitchell L

    2005-07-01

    Organisms growing in acidic environments, pH<3, would be expected to possess fundamentally different molecular structures and physiological controls in comparison with similar species restricted to neutral pH. We begin to investigate this premise by determining the magnitude of the transmembrane electrochemical H+ gradient in an acidophilic Chlamydomonas sp. (ATCC PRA-125) isolated from the Rio Tinto, a heavy metal laden, acidic river (pH 1.7-2.5). This acidophile grows most rapidly at pH 2 but is capable of growth over a wide pH range (1.5-7.0), while Chlamydomonas reinhardtii is restricted to growth at pH>or=3 with optimal growth between pH 5.5 and 8.5. With the fluorescent H+ indicator, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), we show that the acidophilic Chlamydomonas maintains an average cytosolic pH of 6.6 in culture medium at both pH 2 and pH 7 while Chlamydomonas reinhardtii maintains an average cytosolic pH of 7.1 in pH 7 culture medium. The transmembrane electric potential difference of Chlamydomonas sp., measured using intracellular electrodes at both pH 2 and 7, is close to 0 mV, a rare value for plants, animals and protists. The 40,000-fold difference in [H+] could be the result of either active or passive mechanisms. Evidence for active maintenance was detected by monitoring the rate of ATP consumption. At the peak, cells consume about 7% more ATP per second in medium at pH 2 than at pH 7. This increased rate of consumption is sufficient to account for removal of H+ entering the cytosol across a membrane with relatively high permeability to H+ (7x10(-8) cm s-1). Our results indicate that the small increase in the rate of ATP consumption can account for maintenance of the transmembrane H+ gradient without the imposition of cell surface H+ barriers. PMID:15961743

  20. Regulation of intracellular pH in cardiac muscle during cell shrinkage and swelling in anisosmolar solutions.

    PubMed

    Whalley, D W; Hemsworth, P D; Rasmussen, H H

    1994-02-01

    The effect on intracellular pH (pHi) of exposure to solutions of progressively increasing osmolarity from 418 to 620 mosM and to hyposmolar solutions (240 mosM) was examined in guinea pig ventricular muscle using ion-selective microelectrodes. Exposure of tissue to 418 mosM Tyrode solution (100 mM sucrose added) produced an intracellular alkalosis of approximately 0.1 U, whereas exposure to 620 mosM solution (300 mM sucrose added) caused an intracellular acidosis of approximately 0.1 U. The maximal rate of recovery of pHi from acidosis induced by an NH4Cl prepulse increased progressively as extracellular osmolarity was raised from 310 to 620 mosM. This suggests that the acidosis observed at steady state in 620 mosM solution is not due to inhibition of the Na(+)-H+ exchanger. In the presence of 10 microM ryanodine, exposure to 620 mosM solution produced a sustained intracellular alkalosis. We suggest that the decrease in pHi during exposure to 620 mosM solution is due, at least in part, to the acidifying influence of Ca2+ release from the sarcoplasmic reticulum. This decrease in pHi is expected to contribute to the negative inotrop reported in studies of cardiac contractility in markedly hyperosmolar solutions. There was no change in pHi when tissue was exposed to hyposmolar solution. However, the maximal rate of recovery of pHi from acidosis was slower in hyposmolar than in isosmolar solution, despite a concomitant decrease in the intracellular buffer capacity. This suggests that osmotic cell swelling results in inhibition of the sarcolemmal Na(+)-H+ exchanger. PMID:8141367

  1. Effects of 2-methoxyethanol on fetal development, postnatal behavior, and embryonic intracellular pH of rats.

    PubMed

    Nelson, B K; Vorhees, C V; Scott, W J; Hastings, L

    1989-01-01

    The industrial solvent 2-methoxyethanol (2ME) is a reproductive and developmental toxicant when administered by inhalation, gavage, and IP injection. The present research established that this solvent can produce teratogenicity in rats when administered in liquid diet. Groups of 10 Sprague-Dawley rats were given various percentages of 2ME in liquid diet on gestation days 7-18. Day 20 fetuses were examined for visceral or skeletal malformations. Concentrations above 0.025% 2ME (approximately 73 mg/kg/day) produced total embryo-mortality. Cardiovascular malformations were produced at lower levels. The teratogenic no-effect level was 0.006% 2ME (16 mg/kg). In a second experiment, groups of 12 Sprague-Dawley rats were given 0, 0.006 and 0.012% of 2ME as above. Litters were culled to 8 pups, and tested for auditory and tactile startle and conditioned lick suppression, and for performance in figure-8 activity and the Cincinnati water maze on postnatal days 48-65. The high dose of 2ME produced approximately 50% mortality in the offspring and increased the number of errors in the Cincinnati maze. No other behavioral effects were observed at either dose. An interaction study was conducted to determine if simultaneous exposure to 2ME and ethanol would reduce the teratogenicity of 2ME, but no reduction was observed. The hypothesis that 2ME acts by altering embryonic intracellular pH was tested by injecting 0.33 ml/kg of 2ME into rats on gestation day 13, and determining embryonic intracellular pH at 2, 4, 8, and 24 hours thereafter. There was an increase in pH at 4 hours, but not at later time points. Another group of rats was given 2ME along with amiloride, which blocks the sodium/hydrogen antiporter. The combined 2ME-amiloride exposure produced an incidence of cardiovascular malformations in fetuses twice that of 2ME alone. These studies confirmed the structural teratogenicity of 2ME even when given in liquid diet, as it was given for the first time in the present study. At

  2. Basis of antimalarial action: non-weak base effects of chloroquine on acid vesicle pH

    SciTech Connect

    Krogstad, D.J.; Schlesinger, P.H.

    1987-03-01

    Biologically active concentrations of chloroquine increase the pH of the parasite's acid vesicles within 3-5 min. This increase in pH results from two mechanisms, one of which is markedly reduced in chloroquine-resistant parasites. Because chloroquine is a weak base, it increases vesicle pH by that mechanism in chloroquine-susceptible and resistant parasites and mammalian cells (based on its two pKs and on the delta pH between the acid vesicle and the extracellular environment). In chloroquine-susceptible parasites, but not resistant parasites or mammalian cells, chloroquine increases the pH of acid vesicles 700- to 800-fold more than can be accounted for by its properties as a weak base. The increase in acid vesicle pH caused by these non-weak base effects of nanomolar chloroquine in susceptible parasites suggests that chloroquine acts by interfering with acid vesicle functions in the parasite such as the endocytosis and proteolysis of hemoglobin, and the intracellular targeting of lysosomal enzymes. The non-weak base effects of nanomolar chloroquine on parasite vesicle pH are also responsible for its safety because these chloroquine concentrations do not affect mammalian cells.

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

    PubMed

    Søndergaard, Rikke V; Henriksen, Jonas R; Andresen, Thomas L

    2014-12-01

    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 of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks. PMID:25411952

  4. Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO3- cotransport.

    PubMed Central

    Gleeson, D; Smith, N D; Boyer, J L

    1989-01-01

    Using the pH-sensitive dye 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein and a continuously perfused subconfluent hepatocyte monolayer cell culture system, we studied rat hepatocyte intracellular pH (pHi) regulation in the presence (+HCO3-) and absence (-HCO3-) of bicarbonate. Baseline pHi was higher (7.28 +/- 09) in +HCO3- than in -HCO3- (7.16 +/- 0.14). Blocking Na+/H+ exchange with amiloride had no effect on pHi in +HCO3- but caused reversible 0.1-0.2-U acidification in -HCO3- or in +HCO3- after preincubation in the anion transport inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Acute Na+ replacement in +HCO3- alos caused acidification which was amiloride independent but DIDS inhibitible. The recovery of pHi from an intracellular acid load (maximum H+ efflux rate) was 50% higher in +HCO3- than in -HCO3-. Amiloride inhibited H+ effluxmax by 75% in -HCO3- but by only 27% in +HCO3-. The amiloride-independent pHi recovery in +HCO3- was inhibited 50-63% by DIDS and 79% by Na+ replacement but was unaffected by depletion of intracellular Cl-, suggesting that Cl-/HCO3- exchange is not involved. Depolarization of hepatocytes (raising external K+ from 5 to 25 mM) caused reversible 0.05-0.1-U alkalinization, which, however, was neither Na+ nor HCO3- dependent, nor DIDS inhibitible, findings consistent with electroneutral HCO3- transport. We conclude that Na+-HCO3- cotransport, in addition to Na+/H+ exchange, is an important regulator of pHi in rat hepatocytes. PMID:2544626

  5. MATra - Magnet Assisted Transfection: combining nanotechnology and magnetic forces to improve intracellular delivery of nucleic acids.

    PubMed

    Bertram, J

    2006-08-01

    Recent efforts combining nanotechnology and magnetic properties resulted in the development and commercialization of magnetic nanoparticles that can be used as carriers for nucleic acids for in vitro transfection and for gene therapy approaches including DNA-based vaccination strategies. The efficiency of intracellular delivery is still a limiting factor for basic cell biological research and also for emerging technologies such as temporary gene silencing based on inhibitory RNA/siRNA. Nanotechnology has resulted in a variety of different nanostructures and especially nanoparticles as carriers in a wide range of new drug delivery systems for conventional drugs, recombinant proteins, vaccines and more recently nucleic acids. It is possible to combine superparamagnetic nanoparticles with magnetic forces to increase, direct and optimize intracellular delivery of biomolecules. This article discusses the main approaches in the field of magnet assisted transfection (MATra) focusing on the transfection or intracellular delivery of nucleic acids, although also suitable to improve the intracellular delivery of other biomolecules. PMID:16918404

  6. Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells 1

    PubMed Central

    Snedden, Wayne A.; Chung, Induk; Pauls, Randy H.; Bown, Alan W.

    1992-01-01

    Addition of l-[U-14C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-14C]glutamate, and (c) efflux of [14C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H+/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H+/l-Glu symport may involve both H+ consumption during 4-aminobutyrate production and ATP-driven H+ efflux. PMID:16668938

  7. Intracellular pH in Gastric and Rectal Tissue Post Cardiac Arrest

    NASA Astrophysics Data System (ADS)

    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.

  8. Effects of chronic pH 6.6 on growth, intracellular pH, and response to 42.0 degrees C hyperthermia of Chinese hamster ovary cells.

    PubMed

    Cook, J A; Fox, M H

    1988-05-01

    Culturing Chinese hamster ovary cells in low pH (6.6) medium for several months altered the reproductive survival of these cells to combined low pH treatments and 42.0 degrees C heating. We isolated new pH-resistant cells (identified as pHV-2) with enhanced ability to grow and divide under a low pH (6.6) environment. Their growth characteristics include (a) a plating efficiency of 70%, (b) a doubling time of 16 to 17 h, and (c) a steady state intracellular pH 0.12 pH units higher than for cells grown at a normal pH of 7.3. The pHV-2 cells had 100- to 200-fold increases in survival after 5 h of heating compared to cells incubated at low pH (6.6) for 4 h prior to and during the heat treatments. In addition, they developed a significant degree of thermotolerance. We measured a progressive decline in the intracellular pH as a function of time at 42.0 degrees C. However, the decrease in the intracellular pH did not seem to be correlated with the increased heat sensitivity. The ability to select for low pH variants may have important implications in the extrapolation of in vitro hyperthermic data to the in vivo situation. PMID:3356006

  9. Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster.

    PubMed Central

    Drummond, I A; McClure, S A; Poenie, M; Tsien, R Y; Steinhardt, R A

    1986-01-01

    Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response. Images PMID:3097504

  10. Long-term effects of thyroid stimulating hormone and insulin on intracellular pH in FRTL-5 cells.

    PubMed

    Wood, A M; Bidey, S P; Soden, J; Robertson, W R

    1992-05-01

    We have studied the chronic effects of TSH (100 microU/ml) and insulin (10 micrograms/ml) on intracellular pH (pH(i)) in FRTL-5 cells using the pH sensitive probe 2'7-bis (2-carboxyethyl-5'-6') carboxyfluorescein. FRTL-5 cells were cultured on Petri dishes either in the presence of 4H, ie. Coons F-12 containing cortisol (10 nM), transferrin (0.5 microgram/ml), glycyl-histidyl lysine acetate (10 ng/ml) and somatostatin (10 micrograms/ml), or with 4H + insulin (5H), 4H + TSH, or 4H + TSH + insulin (6H). pH(i) was measured in small groups of cells by microspectrofluorimetry both in the presence and absence of bicarbonate ions after cells had been deprived of serum for at least a day. In the absence of TSH, insulin and bicarbonate ions, pH(i) was 7.26 +/- 0.18 (mean +/- SD, n = 49) rising to 7.89 +/- 0.09 (n = 59) and 7.43 +/- 0.1 (n = 55) in the presence of TSH (4H + TSH) and insulin (5H) respectively. Addition of both insulin and TSH (6H) resulted in a pH(i) of 7.75 +/- 0.09 (n = 40). In the absence of TSH and insulin, but the presence of bicarbonate ions, pH(i) was 7.29 +/- 0.12 (mean +/- SD n = 47) rising to 7.72 +/- 0.07 (n = 59) in 4H + TSH and 7.48 +/- 0.08 (n = 60) in 5H. pH(i) in the presence of both TSH and insulin was 7.81 +/- 0.03 (n = 60). In conclusion, both insulin and TSH caused an intracellular alkalinization, TSH markedly so, even in the presence of bicarbonate ions. PMID:1613417

  11. Asterosap-induced elevation in intracellular pH is indispensable for ARIS-induced sustained increase in intracellular Ca2+ and following acrosome reaction in starfish spermatozoa.

    PubMed

    Kawase, Osamu; Minakata, Hiroyuki; Hoshi, Motonori; Matsumoto, Midori

    2005-02-01

    In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, namely ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for the induction of acrosome reaction. For the induction, ARIS alone is enough in high-Ca2+ or high-pH seawater, but, besides ARIS, the addition of either Co-ARIS or asterosap is required in normal seawater. Asterosap transiently increased both the intracellular pH (pHi) and Ca2+ ([Ca2+]i), while ARIS slightly elevated the basal level of [Ca2+]i. However, a sustained elevation of [Ca2+]i and acrosome reaction occurred if sperm were simultaneously treated with ARIS and asterosap. EGTA inhibited the sustained [Ca2+]i elevation and acrosome reaction. The sustained [Ca2+]i elevation and acrosome reaction were highly susceptible to SKF96365 and Ni2+, specific blockers of the store-operated Ca2+ channel (SOC). These results suggest that sustained [Ca2+]i elevation, mediated by the SOC-like channel, is a prerequisite for the acrosome reaction. In high-pH seawater, ARIS alone induced a prominent [Ca2+]i increase and acrosome reaction, which were similarly sensitive to SKF96365. The acrosome reaction was effectively induced by ARIS alone when pHi was artificially increased to more than 7.7. Asterosap increased pHi from 7.6 +/- 0.1 to 7.7 +/- 0.1. Furthermore, the sustained [Ca2+]i elevation and acrosome reaction, induced by a combination of ARIS and asterosap, were drastically inhibited by a slight reduction in pHi. Taking these results into account, we suggest that an asterosap-induced pHi elevation is required for triggering the ARIS-induced sustained [Ca2+]i elevation and consequent acrosome reaction. PMID:15984164

  12. The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones.

    PubMed Central

    Thomas, R C

    1976-01-01

    1. Intracellular pH (pHi) was measured using pH-sensitive glass micro-electrodes. The effects on pHi of CO2 applied externally and HCO3-, H+ and NH4+ injected iontophoretically, were investigated. 2. The transport numbers for iontophoretic injection into aqueous micro-droples were found by potentiometric titration to be 0-3 for HCO3- and 0-94 for H+. 3. Exposure to Ringer, pH 7-5, equilibrated with 2-2% CO2 caused a rapid, but only transient, fall in pHi. Within 1 or 2 min pHi began to return exponentially to normal, with a time constant of about 5 min. 4. When external CO2 was removed, pHi rapidly increased, and then slowly returned to normal. The pHi changes with CO2 application or removal gave a calculated intracellular buffer value of about 30 m-equiv H+/pH unit per litre. 5. Injection of HCO3- caused a rise in pHi very similar to that seen on removal of external CO2. 6. The pHi responses to CO2 application, CO2 removal and HCO3- injection were slowed by the carbonic anhydrase inhibitor acetazolamide. 7. H+ injection caused a transient fall in pHi. In CO2 Ringer pHi fell less and recovered faster than in CO2-free Ringer. Calculation of the internal buffer value from the pHi responses to H+ and HCO3- injection gave very similar values. 8. The internal buffer value (measured by H+ injection) was greatly increased by exposure to CO2 Ringer. Acetazolamide reduced this effect of CO2, suggesting that the function of intracellular carbonic anhydrase may be to maximize the internal buffering power in CO2. 9. It was concluded that the internal HCO3- was determined primarily by the CO2 level and pHi, that internal HCO3- made a large contribution to the buffering power, and that after internal acidfication pHi was restored to normal by active transport of H+, OH- or HCO3- across the cell membrane. The active transport was much faster in CO2 than in CO2-free Ringer. PMID:4614

  13. Genetic interactions among the Arl1 GTPase and intracellular Na(+) /H(+) antiporters in pH homeostasis and cation detoxification.

    PubMed

    Marešová, Lydie; Sychrová, Hana

    2010-11-01

    The roles of intracellular GTPase Arl1 and organellar cation/H(+) antiporters (Kha1 and Nhx1) in Saccharomyces cerevisiae tolerance to various stress factors were investigated and interesting new phenotypes of strains devoid of these proteins were found. The role of Arl1 GTPase in their tolerance to various cations is not caused by an altered plasma-membrane potential. Besides the known sensitivity of arl1 mutants to high temperature, we discovered their sensitivity to low temperature. We found for the first time that in the absence of Arl1p, Kha1p increases potassium, sodium and lithium tolerance, and can thus be categorized as an antiporter with broad substrate specificity. Kha1p also participates in the detoxification of undesired chemical compounds, pH regulation and growth at nonoptimal temperatures. Cells with the combined deletions of all three genes have considerable difficulty growing under nonoptimal conditions. We conclude that Arl1p, Kha1p and Nhx1p collaborate in survival strategies at nonoptimal pH, temperatures and cation concentrations, but work independent of each other. PMID:20659170

  14. Effects of micro electric current load during cooling of plant tissues on intracellular ice crystal formation behavior and pH.

    PubMed

    Ninagawa, Takako; Kawamura, Yukio; Konishi, Tadashi; Narumi, Akira

    2016-08-01

    Cryopreservation techniques are expected to evolve further to preserve biomaterials and foods in a fresh state for extended periods of time. Long-term cryopreservation of living materials such as food and biological tissue is generally achieved by freezing; thus, intracellular freezing occurs. Intracellular freezing injures the cells and leads to cell death. Therefore, a dream cryopreservation technique would preserve the living materials without internal ice crystal formation at a temperature low enough to prevent bacterial activity. This study was performed to investigate the effect of micro electrical current loading during cooling as a new cryopreservation technique. The behavior of intracellular ice crystal formation in plant tissues with or without an electric current load was evaluated using the degree of supercooling, degree of cell deformation, and grain size and growing rate of intracellular ice crystal. Moreover, the transition of intracellular pH during plant tissue cooling with or without electric current loading was also examined using the fluorescence intensity ratio to comprehend cell activity at lower temperatures. The results indicated that micro electric current load did not only decrease the degree of cell deformation and grain size of intracellular ice crystal but also reduced the decline in intracellular pH due to temperature lowering, compared with tissues subjected to the same cooling rate without an electric current load. Thus, the effect of electric current load on cryopreservation and the potential of a new cryopreservation technique using electric current load were discussed based on these results. PMID:27343137

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

    PubMed Central

    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. PMID:26375462

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

    PubMed

    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. PMID:26375462

  17. Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

    PubMed Central

    Gesbert, Gael; Ramond, Elodie; Tros, Fabiola; Dairou, Julien; Frapy, Eric; Barel, Monique

    2014-01-01

    Intracellular bacterial pathogens have adapted their metabolism to optimally utilize the nutrients available in infected host cells. We recently reported the identification of an asparagine transporter required specifically for cytosolic multiplication of Francisella. In the present work, we characterized a new member of the major super family (MSF) of transporters, involved in isoleucine uptake. We show that this transporter (here designated IleP) plays a critical role in intracellular metabolic adaptation of Francisella. Inactivation of IleP severely impaired intracellular F. tularensis subsp. novicida multiplication in all cell types tested and reduced bacterial virulence in the mouse model. To further establish the importance of the ileP gene in F. tularensis pathogenesis, we constructed a chromosomal deletion mutant of ileP (ΔFTL_1803) in the F. tularensis subsp. holarctica live vaccine strain (LVS). Inactivation of IleP in the F. tularensis LVS provoked comparable intracellular growth defects, confirming the critical role of this transporter in isoleucine uptake. The data presented establish, for the first time, the importance of isoleucine utilization for efficient phagosomal escape and cytosolic multiplication of Francisella and suggest that virulent F. tularensis subspecies have lost their branched-chain amino acid biosynthetic pathways and rely exclusively on dedicated uptake systems. This loss of function is likely to reflect an evolution toward a predominantly intracellular life style of the pathogen. Amino acid transporters should be thus considered major players in the adaptation of intracellular pathogens. PMID:25332124

  18. Intracellular release of rapamycin from poly (lactic acid) nanospheres modifies autophagy.

    PubMed

    Nagata, Junpei; Matsui, Makoto; Tabata, Yasuhiko

    2016-09-01

    The objective of this study is to investigate the autophagy activity of cells by the intracellular release of rapamycin (Rapa) of an autophagy inducer. Rapa was incorporated into nanospheres of poly (lactic-co-glycolic acid) (PLGA) for the controlled release of Rapa. Rapa was released from the PLGA nanospheres incorporating rapamycin (Rapa-PLGA-NS) with time while the Rapa-PLGA-NS were hydrolytically degraded. When human hepatocellular carcinoma (HepG2) cells were incubated with the Rapa-PLGA-NS, the Rapa-PLGA-NS were internalized, and the intracellular concentration was maintained over four days, indicating the intracellular Rapa release. The microtubule-associated protein 1 light chain (LC3) of an autophagy marker was significantly high for the Rapa-PLGA-NS group compared with the free Rapa group even after four days incubation. In addition, intracellular harmful ubiquitinated proteins were degraded by the intracellular release of Rapa even after four days incubation in contrast to free Rapa. It is concluded that the intracellular Rapa release is effective in modulating the autophagy activity over a longer time period. PMID:27320771

  19. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

    SciTech Connect

    Galkina, Svetlana I. . E-mail: galkina@genebee.msu.su; Sud'ina, Galina F.; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na{sup +} and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl{sup -} efflux through chloride channels and Na{sup +} influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  20. Intracellular pH and its relationship to regulation of ion transport in normal and cystic fibrosis human nasal epithelia.

    PubMed Central

    Willumsen, N J; Boucher, R C

    1992-01-01

    1. Intracellular pH (pHi) of cultured human airway epithelial cells from normal and cystic fibrosis (CF) subjects were measured with double-barrelled pH-sensitive liquid exchanger microelectrodes. The cells, which were grown to confluence on a permeable collagen matrix support, were mounted in a modified miniature Ussing chamber. All studies were conducted under open circuit conditions. Values are given as means +/- S.E.M. and n refers to the number of preparations. 2. Normal preparations (n = 15) were characterized by a transepithelial potential difference (Vt) of -18 +/- 2 mV, an apical membrane potential (Va) of -19 +/- 2 mV, a basolateral membrane potential (Vb) of -37 +/- 2 mV, a transepithelial resistance (Rt) of 253 +/- 15 omega cm2, a fractional apical membrane resistance (fRa) of 0.40 +/- 0.04 and an equivalent short circuit current (Ieq) of -73 +/- 7 microA cm-2. 3. CF preparations (n = 13) were characterized by a Vt of -46 +/- 7 mV, a Va of 3 +/- 5 mV, a Vb of -43 +/- 3 mV, Rt of 373 +/- 47 omega cm2, fRa of 0.44 +/- 0.04 and an Ieq of -130 +/- 16 microA cm-2. All parameters except Vb and fRa were significantly different (P < 0.025) from those of normal preparations. 4. Despite large differences in electrochemical driving force for proton flow across the apical cell membranes between normal and CF preparations (-4 +/- 3 mV and 20 +/- 7 mV, respectively), pHi was similar (7.15 +/- 0.02 and 7.11 +/- 0.05, respectively). The driving force across the basolateral membrane was similar in normal and CF preparations (22 +/- 3 and 26 +/- 3 mV, respectively). 5. Intracellular alkalinization achieved by removal of CO2 from the luminal Ringer solution or by luminal ammonium prepulse led to stimulation of Ieq in both normal (from -58 to -70 microA cm-2, n = 4; P < 0.05) and CF (from -144 to -163 microA cm-2, n = 4; P < 0.005) preparations. The increase in Ieq was associated with a reduction of Rt, increase in fRa, and hyperpolarization of Vb. All changes in

  1. Soil sorption of acidic pesticides: modeling pH effects.

    PubMed

    Spadotto, Claudio A; Hornsby, Arthur G

    2003-01-01

    A model of acidic pesticide sorption in soils was developed from theoretical modeling and experimental data, which initially considered a combination of a strongly acidic pesticide and a variable-charge soil with high clay content. Contribution of 2,4-D [(2,4-dichlorophenoxy) acetic acid] anionic-form sorption was small when compared with molecular sorption. Dissociation of 2,4-D was not sufficient to explain the variation in Kd as a function of pH. Accessibility of soil organic functional groups able to interact with the pesticide (conformational changes) as a function of organic matter dissociation was proposed to explain the observed differences in sorption. Experimental 2,4-D sorption data and K(oc) values from literature for flumetsulam [N-(2,6-difluorophenyl)-5-methyl [1,2,4] triazolo [1,5-a] pyrimidine-2-sulfonamide] and sulfentrazone [N-[2,4-dichloro-5-[4-(difluromethyl)-4,5-dihydro-3-methyl-5-oxo-1H-1,2,4-triazol-1-yl] phenyl] methanesulfonamide] in several soils fit the model. PMID:12809295

  2. Hyaluronic Acid-Based Nanocarriers for Intracellular Targeting: Interfacial Interactions with Proteins in Cancer

    PubMed Central

    Choi, Ki Young; Saravanakumar, Gurusamy; Park, Jae Hyung; Park, Kinam

    2011-01-01

    The therapeutic efficacy of most drugs is greatly depends on their ability to cross the cellular barrier and reach their intracellular target sites. To transport the drugs effectively through the cellular membrane and to deliver them into the intracellular environment, several interesting smart carrier systems based on both synthetic or natural polymers have been designed and developed. In recent years, hyaluronic acid (HA) has emerged as a promising candidate for intracellular delivery of various therapeutic and imaging agents because of its innate ability to recognize specific cellular receptors that overexpressed on diseased cells. The aim of this review is to highlight the significance of HA in cancer, and to explore the recent advances of HA-based drug carriers towards cancer imaging and therapeutics. PMID:22079699

  3. Effects of varying media, temperature, and growth rates on the intracellular concentrations of yeast amino acids.

    PubMed

    Martínez-Force, E; Benítez, T

    1995-01-01

    Variations of the yeast free amino acid pool under different culture conditions were studied in two Saccharomyces strains, the laboratory haploid strain S288C and the industrial fermentative yeast IFI256. The internal amino acid pool of both strains was measured when grown in laboratory (minimal and complete) versus semiindustrial (molasses with or without added biotin and/or diammonium phosphate) media, in fermentable (glucose, fructose, sucrose) versus respirable (glycerol) carbon sources, in different temperatures (22, 30, and 37 degrees C), pHs (2.0-4.75), and growth rates (0.018-0.24 h-1) in continuous culture, and at different phases of the growth curve in batch culture (lag, exponential, early and late stationary). Results indicated that environmental conditions, particularly the presence of amino acids in the media, enormously influenced the intracellular amino acid concentration. Higher values were detected in molasses than in laboratory media and in fermentable carbon sources (glucose, fructose, sucrose) than in glycerol. Variations in the amino acid pool along the growth curve were greater at 37 degrees C than at other temperatures; in all cases, the highest values were measured at the beginning of the exponential phase. In continuous culture and at different growth rates, intracellular free amino acid concentrations increased by 3-10-fold when the growth rate was lower than 0.05 h-1, representing 20-35% of the total (free plus protein) amino acid content and indicating that amino acid yield was a partly growth-linked parameter. PMID:7654310

  4. Intracellular Localization of the Neurotoxin 2,4-Diaminobutyric Acid in Lathyrus sylvestris L. Leaf Tissue.

    PubMed

    Foster, J G; Cress, W D; Wright, S F; Hess, J L

    1987-04-01

    The intracellular distribution of the neurotoxin 2,4-diaminobutyric acid (DABA) in mature leaves of the perennial legume Lathyrus sylvestris L. var ;Lathco' (flatpea) was determined using subcellular fractions from mesophyll protoplasts. Chloroplasts contained about 15% of the cellular DABA. At least 75% of the DABA was vacuolar, based on the assumptions that each protoplast contained a single vacuole and that acid phosphatase occurred exclusively in the vacuole. DABA was not detectable in peroxisomal and mitochondrial fractions. Because the vacuole is not a major site of amino acid synthesis, this distribution implicates synthesis of DABA within chloroplasts with subsequent transport to and storage within the vacuoles of the mesophyll cells. PMID:16665360

  5. Amino Acid Sensing by mTORC1: Intracellular Transporters Mark the Spot.

    PubMed

    Goberdhan, Deborah C I; Wilson, Clive; Harris, Adrian L

    2016-04-12

    Cell metabolism and growth are matched to nutrient availability via the amino-acid-regulated mechanistic target of rapamycin complex 1 (mTORC1). Transporters have emerged as important amino acid sensors controlling mTOR recruitment and activation at the surface of multiple intracellular compartments. Classically, this has involved late endosomes and lysosomes, but now, in a recent twist, also the Golgi apparatus. Here we propose a model in which specific amino acids in assorted compartments activate different mTORC1 complexes, which may have distinct drug sensitivities and functions. We will discuss the implications of this for mTORC1 function in health and disease. PMID:27076075

  6. Effects of pH adjustment and sodium ions on sour taste intensity of organic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protonated organic acid species have been shown to be the primary stimuli responsible for sour taste of organic acids. However, we have observed that sour taste may be modulated when the pH of acid solutions is raised using sodium hydroxide. Objectives were to evaluate the effect of pH adjustment on...

  7. Interpretation of pH, acidity, and alkalinity in fisheries and aquaculture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Measurements of pH, acidity, and alkalinity are commonly used to describe water quality. The three variables are interrelated and are sometimes confused. The pH of water is an intensity factor, while the acidity and alkalinity of waters are capacity factors. More precisely, acidity and alkalinity ar...

  8. Glycoprotein B of Herpes Simplex Virus 2 Has More than One Intracellular Conformation and Is Altered by Low pH

    PubMed Central

    2012-01-01

    The crystal structure of herpes simplex virus (HSV) gB identifies it as a class III fusion protein, and comparison with other such proteins suggests this is the postfusion rather than prefusion conformation, although this is not proven. Other class III proteins undergo a pH-dependent switch between pre- and postfusion conformations, and a low pH requirement for HSV entry into some cell types suggests that this may also be true for gB. Both gB and gH undergo structural changes at low pH, but there is debate about the extent and significance of the changes in gB, possibly due to the use of different soluble forms of the protein and different assays for antigenic changes. In this study, a complementary approach was taken, examining the conformations of full-length intracellular gB by quantitative confocal microscopy with a panel of 26 antibodies. Three conformations were distinguished, and low pH was found to be a major influence. Comparison with previous studies indicates that the intracellular conformation in low-pH environments may be the same as that of the soluble form known as s-gB at low pH. Interestingly, the antibodies whose binding was most affected by low pH both have neutralizing activity and consequently must block either the function of a neutral pH conformation or its switch from an inactive form to an activated form. If one of the intracellular conformations is the fusion-active form, another factor required for fusion is presumably absent from wherever that conformation is present in infected cells so that inappropriate fusion is avoided. PMID:22514344

  9. Therapeutic potential of analogues of amiloride: inhibition of the regulation of intracellular pH as a possible mechanism of tumour selective therapy.

    PubMed Central

    Maidorn, R. P.; Cragoe, E. J.; Tannock, I. F.

    1993-01-01

    The extracellular pH (pHe) in solid tumours is frequently lower than the pHe in normal tissues. Cells within an acidic environment depend on mechanisms which regulate intracellular pH (pHi) for their survival, including the Na+/H+ antiport which exports protons in exchange for Na+ ions. Amiloride and its analogues DMA (5-(N,N-dimethyl)amiloride), MIBA (5-(N-methyl-N-isobutyl)amiloride) and EIPA (5-(N-ethyl-N-isopropyl)amiloride) are known to inhibit the Na+/H+ antiport and therefore decrease the cells ability to regulate pHi. All three analogues were found to be potent inhibitors of the antiport in human MGH-U1 and murine EMT-6 cells, with DMA being approximately 20, MIBA 100 and EIPA 200-fold as potent as amiloride; EIPA also gave more complete suppression of the Na+/H+ antiport. These agents were not toxic to cells when used alone; however, in combination with nigericin, an agent which acidifies cells, all three analogues were toxic to cells at pHe < 7.0, and markedly enhanced the toxicity of nigericin alone. Cell killing was greatest for nigericin used with EIPA or MIBA. None of the agents were toxic to cells at pHe 7.0 or above. When used against variant cells lacking the Na+/H+ antiport (PS-120 cells) EIPA did not enhance the cytotoxicity of nigericin alone, suggesting that the observed effect was due to inhibition of Na+/H+ exchange, rather than due to non-specific effects. The combination of EIPA and nigericin gave similar cell killing in previously dissociated and intact MGH-U1 spheroids, suggesting that the agents have good penetration of solid tissue. Preliminary experiments using EMT-6 tumours in mice suggested that EIPA and nigericin were able to enhance the toxicity of radiation in vivo, presumably through selective effects against the hypoxic (and probably acidic) subpopulation of cells that is resistant to radiation. PMID:8381657

  10. Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signaling

    PubMed Central

    Patel, Sandip; Docampo, Roberto

    2010-01-01

    Changes in cytosolic calcium concentration are crucial for a variety of cellular processes in all cells. It has long been appreciated that calcium is stored and released from intracellular calcium stores such as the endoplasmic reticulum. However, emerging evidence indicates that calcium is also dynamically regulated by a seemingly disparate collection of acidic organelles. Here, we review the defining features of these acidic calcium stores and highlight recent progress in understanding the mechanisms of uptake and release of calcium from these stores. We also examine the nature of calcium buffering within the stores and summarize the physiological and patho-physiological significance of these ubiquitous organelles in calcium signaling. PMID:20303271

  11. The PH gene determines fruit acidity and contributes to the evolution of sweet melons.

    PubMed

    Cohen, Shahar; Itkin, Maxim; Yeselson, Yelena; Tzuri, Galil; Portnoy, Vitaly; Harel-Baja, Rotem; Lev, Shery; Sa'ar, Uzi; Davidovitz-Rikanati, Rachel; Baranes, Nadine; Bar, Einat; Wolf, Dalia; Petreikov, Marina; Shen, Shmuel; Ben-Dor, Shifra; Rogachev, Ilana; Aharoni, Asaph; Ast, Tslil; Schuldiner, Maya; Belausov, Eduard; Eshed, Ravit; Ophir, Ron; Sherman, Amir; Frei, Benedikt; Neuhaus, H Ekkehard; Xu, Yimin; Fei, Zhangjun; Giovannoni, Jim; Lewinsohn, Efraim; Tadmor, Yaakov; Paris, Harry S; Katzir, Nurit; Burger, Yosef; Schaffer, Arthur A

    2014-01-01

    Taste has been the subject of human selection in the evolution of agricultural crops, and acidity is one of the three major components of fleshy fruit taste, together with sugars and volatile flavour compounds. We identify a family of plant-specific genes with a major effect on fruit acidity by map-based cloning of C. melo PH gene (CmPH) from melon, Cucumis melo taking advantage of the novel natural genetic variation for both high and low fruit acidity in this species. Functional silencing of orthologous PH genes in two distantly related plant families, cucumber and tomato, produced low-acid, bland tasting fruit, showing that PH genes control fruit acidity across plant families. A four amino-acid duplication in CmPH distinguishes between primitive acidic varieties and modern dessert melons. This fortuitous mutation served as a preadaptive antecedent to the development of sweet melon cultigens in Central Asia over 1,000 years ago. PMID:24898284

  12. Intracellular domoic acid production in Pseudo-nitzschia multistriata isolated from the Gulf of Naples (Tyrrhenian Sea, Italy).

    PubMed

    Amato, Alberto; Lüdeking, Alexander; Kooistra, Wiebe H C F

    2010-01-01

    Twenty-six Pseudo-nitzschia multistriata cultures were tested for intracellular domoic acid production and fourteen were found to be toxic. Four suboptimal growth conditions were compared with conditions observed to be optimal to explore possible triggers for intracellular domoic acid production. Silica- and phosphate-limitation and low light treatment induced elevated toxin concentrations whereas high temperature appeared to suppress it. Inheritance of the toxin-production ability was investigated by measuring intracellular toxin content in a total of thirty-nine F(1) strains from two different crosses. Results showed radical differences in domoic acid levels among the F(1) offspring from the same parents. PMID:19615395

  13. Quantitative Intracellular Localization of Cationic Lipid-Nucleic Acid Nanoparticles with Fluorescence Microscopy.

    PubMed

    Majzoub, Ramsey N; Ewert, Kai K; Safinya, Cyrus R

    2016-01-01

    Current activity in developing synthetic carriers of nucleic acids (NA) and small molecule drugs for therapeutic applications is unprecedented. One promising class of synthetic vectors for the delivery of therapeutic NA is PEGylated cationic liposome (CL)-NA nanoparticles (NPs). Chemically modified PEG-lipids can be used to surface-functionalize lipid-NA nanoparticles, allowing researchers to design active nanoparticles that can overcome the various intracellular and extracellular barriers to efficient delivery. Optimization of these functionalized vectors requires a comprehensive understanding of their intracellular pathways. In this chapter we present two distinct methods for investigating the intracellular activity of PEGylated CL-NA NPs using quantitative analysis with fluorescence microscopy.The first method, spatial localization, describes how to prepare fluorescently labeled CL-NA NPs, perform fluorescence microscopy and properly analyze the data to measure the intracellular distribution of nanoparticles and fluorescent signal. We provide software which allows data from multiple cells to be averaged together and yield statistically significant results. The second method, fluorescence colocalization, describes how to label endocytic organelles via Rab-GFPs and generate micrographs for software-assisted NP-endocytic marker colocalization measurements. These tools will allow researchers to study the endosomal trafficking of CL-NA NPs which can guide their design and improve their efficiency. PMID:27436314

  14. Intracellular pH measurements of the whole head and the basal ganglia in chronic liver disease: a phosphorus-31 MR spectroscopy study.

    PubMed

    Patel, N; Forton, D M; Coutts, G A; Thomas, H C; Taylor-Robinson, S D

    2000-09-01

    The purpose of this study was to determine the intracellular pH of the whole head and in voxels localized to the basal ganglia in patients with chronic liver disease using phosphorus-31 magnetic resonance spectroscopy (31P MRS). The study group compromised 82 patients with biopsy-proven cirrhosis (43 Child's grade A, 25 Child's grade B and 14 Child's grade C). Eleven subjects showed no evidence of neuropsychiatric impairment on clinical, psychometric and electrophysiological testing, 37 showed evidence of minimal hepatic encephalopathy and 34 had overt hepatic encephalopathy. Unlocalized 31P MRS of the whole head was performed in 48 patients and 10 healthy volunteers. Localized 31P MRS of the basal ganglia was performed in the 34 patients and in 20 healthy volunteers. The intracellular pH values were calculated from the chemical shift difference between the inorganic phosphate (P) and phosphocreatine (PCr) resonances. The percentage inorganic phosphate (%Pi), phosphocreatine (%PCr) and betaNTP signals, relative to the total 31P signal, and peak area ratios of inorganic phosphate and phosphocreatine, relative to betaNTP were also measured. There were no differences between patients and volunteers in intracellular pH in 31P MR spectra measured from the whole head or the basal ganglia. There was no correlation between the severity of encephalopathy (West Haven criteria) or liver dysfunction (Child score) and intracellular pH values. There was also no significant change in the inorganic phosphate, phosphocreatine or betaNTP resonances in spectra acquired from the whole head. However, in spectra localized to the basal ganglia, there was a significant increase in mean P/NTP (p=0.02) and PCr/NTP (p=0.009). The mean %Pi and mean %PCr were also increased (p=0.06; p=0.05, respectively), but there was no significant change in mean %betaNTP. When the patient population was classified according to the severity of encephalopathy, those with overt disease had a higher mean P

  15. 2,3-Butanediol fermentation promotes growth of Serratia plymuthica at low pH but not survival of extreme acid challenge.

    PubMed

    Vivijs, Bram; Moons, Pieter; Geeraerd, Annemie H; Aertsen, Abram; Michiels, Chris W

    2014-04-01

    The mechanisms by which Enterobacteriaceae can survive or grow at low pH are of interest because members of this family are increasingly linked to problems of spoilage and foodborne infection related to mildly acidic foods. In this work, we investigated the contribution of the 2,3-butanediol fermentation pathway in coping with specific forms of acid stress in Serratia plymuthica RVH1. This pathway consumes intracellular protons, similar to the amino acid decarboxylases which are involved in acid resistance in Enterobacteriaceae. While its role in preventing excessive acidification in media with an initial neutral pH but containing fermentable sugars has been established, we here addressed the question whether it supports survival of severe acid challenge (pH2.5-3.5) and/or enhances the ability to initiate growth at moderately low pH (pH4.0-5.0) in acidified LB medium and in tomato juice. Using a budAB::cat mutant, deficient in 2,3-butanediol fermentation, we showed that the pathway did not influence survival in simulated gastric fluid and is not involved in the acid tolerance response (ATR) in S. plymuthica RVH1. On the other hand, the pathway promoted growth at moderately low pH. In acidified LB medium, the mutant stopped growing at a lower final cell density than the wild-type strain. In tomato juice, additionally, the minimal pH at which the mutant could grow (pH4.20-4.30) was increased compared to that of the wild-type (pH4.10). Growth of the wild-type strain was often accompanied by a pH increase, in contrast to the budAB::cat mutant, where the opposite was observed. However, the differences in growth between the wild-type and budAB::cat mutant could not only be explained by external pH, suggesting that the 2,3-butanediol fermentation contributed to intracellular pH homeostasis. Based on these data, we propose the contribution to growth at low pH as a novel biological function of 2,3-butanediol fermentation in Enterobacteriaceae. PMID:24531037

  16. Organic Acid Excretion in Penicillium ochrochloron Increases with Ambient pH

    PubMed Central

    Vrabl, Pamela; Fuchs, Viktoria; Pichler, Barbara; Schinagl, Christoph W.; Burgstaller, Wolfgang

    2012-01-01

    Despite being of high biotechnological relevance, many aspects of organic acid excretion in filamentous fungi like the influence of ambient pH are still insufficiently understood. While the excretion of an individual organic acid may peak at a certain pH value, the few available studies investigating a broader range of organic acids indicate that total organic acid excretion rises with increasing external pH. We hypothesized that this phenomenon might be a general response of filamentous fungi to increased ambient pH. If this is the case, the observation should be widely independent of the organism, growth conditions, or experimental design and might therefore be a crucial key point in understanding the function and mechanisms of organic acid excretion in filamentous fungi. In this study we explored this hypothesis using ammonium-limited chemostat cultivations (pH 2–7), and ammonium or phosphate-limited bioreactor batch cultivations (pH 5 and 7). Two strains of Penicillium ochrochloron were investigated differing in the spectrum of excreted organic acids. Confirming our hypothesis, the main result demonstrated that organic acid excretion in P. ochrochloron was enhanced at high external pH levels compared to low pH levels independent of the tested strain, nutrient limitation, and cultivation method. We discuss these findings against the background of three hypotheses explaining organic acid excretion in filamentous fungi, i.e., overflow metabolism, charge balance, and aggressive acidification hypothesis. PMID:22493592

  17. Effect of pH on visualization of fatty acids as myelin figures in mouse adipose tissue by freeze-fracture electron microscopy.

    PubMed

    Amende, L M; Blanchette-Mackie, E J; Chernick, S S; Scow, R O

    1985-10-23

    We studied the effect of pH on visualization of fatty acids as myelin figures in young mouse epididymal adipose tissue. Fatty acid content of the tissue was increased to 12.4 nmol/mg wet weight by treating the tissue with 380 microM isoproterenol at pH 7.4 for 15 min in the absence of glucose and albumin. Myelin figures were found in freeze-fracture replicas of isoproterenol-treated tissue fixed with glutaraldehyde at pH 7.4 and then incubated and glycerinated at pH 8.1. Myelin figures were seen in replicas as concave or convex laminated sheets and long cylindrical multilamellar structures in fat cells and extracellular space. Myelin figures were sometimes seen in cells extending from the surface of intracellular lipid droplets, the site of lipolysis, to the cell surface and extracellular space. Myelin figures were not found in isoproterenol-treated tissue fixed at pH 7.4 and processed at pH 7.0. Smooth-surfaced droplets, instead, were found in these tissues in the extracellular space. Neither myelin figures nor smooth-surfaced droplets were found in tissues treated with insulin and glucose (to reduce fatty acid content to 1.4 nmol/mg), fixed at pH 7.4 and processed at either pH 8.1 or pH 7.0. Lowering pH of the media to 4.5 during processing of tissues treated with isoproterenol at pH 9.0 caused disappearance of myelin figures and appearance of smooth-surfaced droplets in the extracellular space. Myelin figures were found in replicas of tissue treated with isoproterenol for 15 min at pH 7.4, incubated 10 min at pH 8.4, quick-frozen and then freeze-fractured, indicating that formation of myelin figures was not dependent on glutaraldehyde fixation and glycerol infiltration of the tissue. Our findings show that excess fatty acids in adipose tissue can be visualized as myelin figures if the tissue is exposed to pH 8.1-9.0 and maintained at or above pH 7.4, or as smooth-surfaced droplets if the tissue is processed at pH 7.0 or 4.5. We conclude that myelin figures

  18. Influence of the pH on the itaconic acid production with Aspergillus terreus.

    PubMed

    Hevekerl, Antje; Kuenz, Anja; Vorlop, Klaus-Dieter

    2014-12-01

    Itaconic acid is mainly produced with the filamentous fungi Aspergillus terreus. An increase in the pH during the production phase of the cultivation resulted in an increase in the itaconic acid concentration. The pH was raised by a single pH shift ranging from pH 4 to 6 or by a pH control to pH 3. Different lyes can be used for the pH shift, but ammonia solution has proven to be the best, because here the productivity does not drop after the pH shift. The highest itaconic acid concentration of 146 g/L was reached when a pH control to pH 3 was started after 2.1 days of cultivation. This is an increase of 68 % to the cultivation without pH control. When this technique was combined with previously found optimizations, a final itaconic acid concentration of 129 g/L was reached after 4.7 days of cultivation, resulting in a productivity of 1.15 g/L/h. PMID:25213913

  19. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles.

    PubMed

    Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

    2015-01-01

    Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours' exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours' exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP-cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

  20. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

    PubMed Central

    Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

    2015-01-01

    Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

  1. Effect of Acidic pH on Expression of Surface-Associated Proteins of Streptococcus oralis

    PubMed Central

    Wilkins, Joanna C.; Beighton, David; Homer, Karen A.

    2003-01-01

    Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions. PMID:12957916

  2. Rhizosphere pH responses to simulated acid rain as measured with glass microelectrodes

    SciTech Connect

    Conkling, B.L.

    1988-01-01

    The objectives of this study were to develop a useful experimental system for studying the rhizosphere of growing roots, and to investigate the effects of bulk soil pH and foliar acid rain application on the rhizosphere pH of alfalfa, corn and soybeans. First, a study was done to compare soil pH measurements made with a standard glass pH electrode with those made using an antimony (Sb) microelectrode. Because of uncertainty with the Sb microelectrodes' response, glass pH-sensitive microelectrodes were made and tested for rhizosphere pH measurements. The influence of soil water pressure gradients in the range of {minus}10 to {minus}1500 kPa in the proximity of the pH and reference electrodes on pH measurements made with microelectrodes was studied. The effect of foliar acid rain application on the rhizosphere pH of alfalfa, corn, and soybean as a function of soil pH were studied. Alfalfa, corn, and soybean were grown into minirhizotrons containing reformed samples of both Seymour A and Bt soil horizons, and the rhizosphere pH measured. The measured in situ bulk soil pH ranged from 4.9 to 6.2 in the A horizon and from 4.0 to 5.7 in the Bt horizon. Plants received acid or non-acid foliar rain applications. Rhizosphere pH was measured using a glass pH-sensitive microelectrode. Acid rain applications caused foliar damage, but had little effect on the rhizosphere pH. The general trend was for the lateral root pH values to be slightly higher than the main root values.

  3. Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation

    PubMed Central

    Aguilera, Paulina; Marcoleta, Andrés; Lobos-Ruiz, Pablo; Arranz, Rocío; Valpuesta, José M.; Monasterio, Octavio; Lagos, Rosalba

    2016-01-01

    Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although

  4. Negative pH and extremely acidic mine waters from Iron Mountain, California

    USGS Publications Warehouse

    Nordstrom, D.K.; Alpers, C.N.; Ptacek, C.J.; Blowes, D.W.

    2000-01-01

    Extremely acidic mine waters with pH values as low as -3.6, total dissolved metal concentrations as high as 200 g/L, and sulfate concentrations as high as 760 g/L, have been encountered underground in the Richmond Mine at Iron Mountain, CA. These are the most acidic waters known. The pH measurements were obtained by using the Pitzer method to define pH for calibration of glass membrane electrodes. The calibration of pH below 0.5 with glass membrane electrodes becomes strongly nonlinear but is reproducible to a pH as low as -4. Numerous efflorescent minerals were found forming from these acid waters. These extreme acid waters were formed primarily by pyrite oxidation and concentration by evaporation with minor effects from aqueous ferrous iron oxidation and efflorescent mineral formation.

  5. A pH and redox dual stimuli-responsive poly(amino acid) derivative for controlled drug release.

    PubMed

    Gong, Chu; Shan, Meng; Li, Bingqiang; Wu, Guolin

    2016-10-01

    A pH and redox dual stimuli-responsive poly(aspartic acid) derivative for controlled drug release was successfully developed through progressive ring-opening reactions of polysuccinimide (PSI). Polyethylene glycol (PEG) chains were grafted onto the polyaspartamide backbone via redox-responsive disulfide linkages, providing a sheddable shell for the polymeric micelles in a reductive environment. Phenyl groups were introduced into the polyaspartamide backbone via the aminolysis reaction of PSI to serve as the hydrophobic segment of micelles. The polyaspartamide scaffold was also functionalized with N-(3-aminopropyl)-imidazole to obtain the pH-responsiveness manifesting as a swelling of the core of micelles at a low pH. The polymeric micelles with a core-shell nanostructure forming in neutral media exhibited both pH and redox responsive characteristics. Doxorubicin (DOX) as a model drug was encapsulated into the core of micelles through both hydrophobic and π-π interactions between aromatic rings and the DOX-loaded polymeric micelles exhibited accelerated drug release behaviors in an acidic and reductive environment due to the swelling of hydrophobic cores and the shedding of PEG shells. Furthermore, the cytocompability of the polymer and the cytotoxicity of DOX-loaded micelles towards Hela cells under corresponding conditions were evaluated, and the endocytosis of DOX-loaded polymeric micelles and the intracellular drug release from micelles were observed. All obtained data indicated that the micelle was a promising candidate for controlled drug release. PMID:27388968

  6. Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization

    NASA Astrophysics Data System (ADS)

    Chiappini, C.; De Rosa, E.; Martinez, J. O.; Liu, X.; Steele, J.; Stevens, M. M.; Tasciotti, E.

    2015-05-01

    The controlled delivery of nucleic acids to selected tissues remains an inefficient process mired by low transfection efficacy, poor scalability because of varying efficiency with cell type and location, and questionable safety as a result of toxicity issues arising from the typical materials and procedures employed. High efficiency and minimal toxicity in vitro has been shown for intracellular delivery of nuclei acids by using nanoneedles, yet extending these characteristics to in vivo delivery has been difficult, as current interfacing strategies rely on complex equipment or active cell internalization through prolonged interfacing. Here, we show that a tunable array of biodegradable nanoneedles fabricated by metal-assisted chemical etching of silicon can access the cytosol to co-deliver DNA and siRNA with an efficiency greater than 90%, and that in vivo the nanoneedles transfect the VEGF-165 gene, inducing sustained neovascularization and a localized sixfold increase in blood perfusion in a target region of the muscle.

  7. Acid phosphatase activity and intracellular collagen degradation by fibroblasts in vitro.

    PubMed

    Yajima, T

    1986-01-01

    Human gingival fibroblasts were cultured with collagen fibrils. The precise process of collagen phagocytosis and the relationship between acid phosphatase activity and intracellular degradation of collagen were investigated by cytochemical methods at the ultrastructural level. The collagen fibrils were first engulfed at one end by cellular processes, or the cell membrane wrapped itself around the middle of the fibrils. Collagen phagocytosis induced acid phosphatase activity in the fibroblast Golgi-endoplasmic reticulum-lysosome system. By application of the tracer lanthanum, deposits were observed in the intercellular spaces and along the fibrils being phagocytosed. At this stage, primary lysosomes were seen in close proximity to the collagen being engulfed, but no signs of fusion were observed. When the fibrils had been interiorized in whole or in part, they ultimately became enclosed within phagosomes, and no tracer was observed along the interiorized portion of the fibrils. Primary lysosomes then fused with these collagen-containing phagosomes to form phagolysosomes. Collagen degradation occurred within these bodies even though the end of a fibril might have protruded outside of the cell. These results suggest that selective and controlled phagocytosis of collagen and intracellular digestion of it may play a central role in the physiological remodeling and metabolic breakdown of the collagen of connective tissues. PMID:3742560

  8. Disrupting Protein Expression with Peptide Nucleic Acids Reduces Infection by Obligate Intracellular Rickettsia

    PubMed Central

    Pelc, Rebecca S.; McClure, Jennifer C.; Kaur, Simran J.; Sears, Khandra T.; Rahman, M. Sayeedur; Ceraul, Shane M.

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria’s ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria. PMID:25781160

  9. Quantum-dot/dopamine bioconjugates function as redox coupled assemblies for in vitro and intracellular pH sensing

    NASA Astrophysics Data System (ADS)

    Medintz, Igor L.; Stewart, Michael H.; Trammell, Scott A.; Susumu, Kimihiro; Delehanty, James B.; Mei, Bing C.; Melinger, Joseph S.; Blanco-Canosa, Juan B.; Dawson, Philip E.; Mattoussi, Hedi

    2010-08-01

    The use of semiconductor quantum dots (QDs) for bioimaging and sensing has progressively matured over the past decade. QDs are highly sensitive to charge-transfer processes, which can alter their optical properties. Here, we demonstrate that QD-dopamine-peptide bioconjugates can function as charge-transfer coupled pH sensors. Dopamine is normally characterized by two intrinsic redox properties: a Nernstian dependence of formal potential on pH and oxidation of hydroquinone to quinone by O2 at basic pH. We show that the latter quinone can function as an electron acceptor quenching QD photoluminescence in a manner that depends directly on pH. We characterize the pH-dependent QD quenching using both electrochemistry and spectroscopy. QD-dopamine conjugates were also used as pH sensors that measured changes in cytoplasmic pH as cells underwent drug-induced alkalosis. A detailed mechanism describing the QD quenching processes that is consistent with dopamine's inherent redox chemistry is presented.

  10. Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels.

    PubMed

    Skatchkov, Serguei N; Bukauskas, Feliksas F; Benedikt, Jan; Inyushin, Mikhail; Kucheryavykh, Yuriy V

    2015-06-17

    Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell-cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions. PMID:26011388

  11. Intracellular spermine prevents acid-induced uncoupling of Cx43 gap junction channels

    PubMed Central

    Skatchkov, Serguei N.; Bukauskas, Feliksas F.; Benedikt, Jan; Inyushin, Mikhail

    2015-01-01

    Polyamines (PAs), such as spermine and spermidine, modulate the activity of numerous receptors and channels in the central nervous system (CNS) and are stored in glial cells; however, little attention has been paid to their role in the regulation of connexin (Cx)-based gap junction channels. We have previously shown that PAs facilitate diffusion of Lucifer Yellow through astrocytic gap junctions in acute brain slices; therefore, we hypothesized that spermine can regulate Cx43-mediated (as the most abundant Cx in astrocytes) gap junctional communication. We used electrophysiological patch-clamp recording from paired Novikoff cells endogenously expressing Cx43 and HeLaCx43-EGFP transfectants to study pH-dependent modulation of cell–cell coupling in the presence or absence of PAs. Our results showed (i) a higher increase in gap junctional communication at higher concentrations of cytoplasmic spermine, and (ii) that spermine prevented uncoupling of gap junctions at low intracellular pH. Taken together, we conclude that spermine enhances Cx43-mediated gap junctional communication and may preserve neuronal excitability during ischemia and trauma when pH in the brain acidifies. We, therefore, suggest a new role of spermine in the regulation of a Cx43-based network under (patho)physiological conditions. PMID:26011388

  12. Control of Gastric H,K-ATPase Activity by Cations, Voltage and Intracellular pH Analyzed by Voltage Clamp Fluorometry in Xenopus Oocytes

    PubMed Central

    Dürr, Katharina L.; Tavraz, Neslihan N.; Friedrich, Thomas

    2012-01-01

    Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E1P and E2P states and measured Rb+ uptake under various ionic and pH conditions. The steady-state E1P/E2P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb+ uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E1P/E2P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V0.5, the voltage, at which the E1P/E2P ratio is 50∶50, by −100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E1P→E2P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb+ uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E1P→E2P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na+ profoundly alters the voltage-dependent E1P/E2P distribution indicating that Na+ ions can act as surrogates for protons regarding the E2P→E1P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting

  13. Cell nucleus targeting for living cell extraction of nucleic acid associated proteins with intracellular nanoprobes of magnetic carbon nanotubes.

    PubMed

    Zhang, Yi; Hu, Zhengyan; Qin, Hongqiang; Liu, Fangjie; Cheng, Kai; Wu, Ren'an; Zou, Hanfa

    2013-08-01

    Since nanoparticles could be ingested by cells naturally and target at a specific cellular location as designed, the extraction of intracellular proteins from living cells for large-scale analysis by nanoprobes seems to be ideally possible. Nucleic acid associated proteins (NAaP) take the crucial position during biological processes in maintaining and regulating gene structure and gene related behaviors, yet there are still challenges during the global investigation of intracellular NAaP, especially from living cells. In this work, a strategy to extract intracellular proteins from living cells with the magnetic carbon nanotube (oMWCNT@Fe3O4) as an intracellular probe is developed, to achieve the high throughput analysis of NAaP from living human hepatoma BEL-7402 cells with a mass spectrometry-based proteomic approach. Due to the specific intracellular localization of the magnetic carbon nanotubes around nuclei and its strong interaction with nucleic acids, the highly efficient extraction was realized for cellular NAaP from living cells, with the capability of identifying 2383 intracellular NAaP from only ca. 10,000 living cells. This method exhibited potential applications in dynamic and in situ analysis of intracellular proteins. PMID:23815738

  14. Primordial soup or vinaigrette: did the RNA world evolve at acidic pH?

    PubMed Central

    2012-01-01

    Background The RNA world concept has wide, though certainly not unanimous, support within the origin-of-life scientific community. One view is that life may have emerged as early as the Hadean Eon 4.3-3.8 billion years ago with an atmosphere of high CO2 producing an acidic ocean of the order of pH 3.5-6. Compatible with this scenario is the intriguing proposal that life arose within alkaline (pH 9-11) deep-sea hydrothermal vents like those of the 'Lost City', with the interface with the acidic ocean creating a proton gradient sufficient to drive the first metabolism. However, RNA is most stable at pH 4-5 and is unstable at alkaline pH, raising the possibility that RNA may have first arisen in the acidic ocean itself (possibly near an acidic hydrothermal vent), acidic volcanic lake or comet pond. As the Hadean Eon progressed, the ocean pH is inferred to have gradually risen to near neutral as atmospheric CO2 levels decreased. Presentation of the hypothesis We propose that RNA is well suited for a world evolving at acidic pH. This is supported by the enhanced stability at acidic pH of not only the RNA phosphodiester bond but also of the aminoacyl-(t)RNA and peptide bonds. Examples of in vitro-selected ribozymes with activities at acid pH have recently been documented. The subsequent transition to a DNA genome could have been partly driven by the gradual rise in ocean pH, since DNA has greater stability than RNA at alkaline pH, but not at acidic pH. Testing the hypothesis We have proposed mechanisms for two key RNA world activities that are compatible with an acidic milieu: (i) non-enzymatic RNA replication of a hemi-protonated cytosine-rich oligonucleotide, and (ii) specific aminoacylation of tRNA/hairpins through triple helix interactions between the helical aminoacyl stem and a single-stranded aminoacylating ribozyme. Implications of the hypothesis Our hypothesis casts doubt on the hypothesis that RNA evolved in the vicinity of alkaline hydrothermal vents. The

  15. Intracellular spermine blocks TRPC4 channel via electrostatic interaction with C-terminal negative amino acids.

    PubMed

    Kim, Jinsung; Moon, Sang Hui; Shin, Young-Cheul; Jeon, Ju-Hong; Park, Kyu Joo; Lee, Kyu Pil; So, Insuk

    2016-04-01

    Transient receptor potential canonical (TRPC) 4 channels are calcium-permeable, nonselective cation channels and are widely expressed in mammalian tissue, especially in the GI tract and brain. TRPC4 channels are known to be involved in neurogenic contraction of ileal smooth muscle cells via generating cationic current after muscarinic stimulation (muscarinic cationic current (mIcat)). Polyamines exist in numerous tissues and are believed to be involved in cell proliferation, differentiation, scar formation, wound healing, and carcinogenesis. Besides, physiological polyamines are essential to maintain inward rectification of cardiac potassium channels (Kir2.1). At membrane potentials more positive than equilibrium potential, intracellular polyamines plug the cytosolic surface of the Kir2.1 so that potassium ions cannot pass through the pore. Recently, it was reported that polyamines inhibit not only cardiac potassium channels but also nonselective cation channels that mediate the generation of mIcat. Here, we report that TRPC4, a definite mIcat mediator, is inhibited by intracellular spermine with great extent. The inhibition was specific to TRPC4 and TRPC5 channels but was not effective to TRPC1/4, TRPC1/5, and TRPC3 channels. For this inhibition to occur, we found that glutamates at 728th and 729th position of TRPC4 channels are essential whereby we conclude that spermine blocks the TRPC4 channel with electrostatic interaction between negative amino acids at the C-terminus of the channel. PMID:26631167

  16. Volatile fatty acids distribution during acidogenesis of algal residues with pH control.

    PubMed

    Li, Yan; Hua, Dongliang; Zhang, Jie; Zhao, Yuxiao; Xu, Haipeng; Liang, Xiaohui; Zhang, Xiaodong

    2013-06-01

    The anaerobic acidification of protein-rich algal residues with pH control (4, 6, 8, 10) was studied in batch reactors, which was operated at mesophilic(35 °C) condition. The distribution of major volatile fatty acids (VFAs) during acidogenesis was emphasized in this paper. The results showed that the acidification efficiency and VFAs distribution in the acid reactor strongly depended on the pH. The main product for all the runs involved acetic acid except that the proportion of butyric acid acidified at pH 6 was relatively higher. The other organic acids remained at lower levels. The VFAs yield reached the maximum value with about 0.6 g VFAs/g volatile solid (VS) added as pH was 8, and also the content of total ammonia nitrogen (TAN) reached the highest values of 9,629 mg/l. Low acidification degrees were obtained under the conditions at pH 4 and 10, which was not suitable for the metabolism of acidogens. Hydralic retention time (HRT) required for different conditions varied. As a consequence, it was indicated that pH was crucial to the acidification efficiency and products distribution. The investigation of acidogenesis process, which was producing the major substrates, short-chain fatty acids, would play the primary role in the efficient operation of methanogenesis. PMID:23381617

  17. Bile Acids Regulate Nuclear Receptor (Nur77) Expression and Intracellular Location to Control Proliferation and Apoptosis

    PubMed Central

    Hu, Ying; Chau, Thinh; Liu, Hui-xin; Liao, Degui; Keane, Ryan; Nie, Yuqiang; Yang, Hui; Wan, Yu-Jui Yvonne

    2014-01-01

    Bile acids (BAs) are endogenous agents capable of causing cancer throughout the gastrointestinal (GI) tract. To uncover the mechanism by which BAs exert carcinogenic effects, both human liver and colon cancer cells as well as mouse primary hepatocytes were treated with BAs and assayed for viability, genotoxic stress, and transcriptional response. BAs induced both Nur77 (NR4A1) and pro-inflammatory gene expression. The intracellular location of BA-induced Nur77 was time-dependent; short-term (1–3 h) exposure induced nuclear Nur77 whereas longer (1–2 days) exposure also increased cytosolic Nur77 expression and apoptosis. Inhibiting Nur77 nuclear export with leptomycin B decreased LCA-induced apoptosis. Extended (7 days) treatment with BA generated resistance to BA with increased nuclear Nur77, viability, and mobility. While, knockdown of Nur77 in BA-resistant cells increased cellular susceptibility to LCA-induced apoptosis. Moreover, in vivo mouse xenograft experiments demonstrated that BA-resistant cells form larger tumors with elevated Nur77 expression compared to parental controls. DNA-binding and gene expression assays identified multiple survival genes (CDK4, CCND2, MAP4K5, STAT5A, and RBBP8) and a pro-apoptosis gene (BID) as Nur77 targets. Consistently, BA-induced up-regulation of the aforementioned genes was abrogated by a lack of Nur77. Importantly, Nur77 was overexpressed in high percentage of human colon and liver cancer specimens and the intracellular location of Nur77 correlated with elevated serum total BA levels in colon cancer patients. These data show for the first time that BAs via Nur77 have a dual role in modulating cell survival and death. Implications: These findings establish a direct link between Nur77 and the carcinogenic effect of bile acids. PMID:25232032

  18. Molecular Dynamics Simulations Capture the Misfolding of the Bovine Prion Protein at Acidic pH

    PubMed Central

    Cheng, Chin Jung; Daggett, Valerie

    2014-01-01

    Bovine spongiform encephalopathy (BSE), or mad cow disease, is a fatal neurodegenerative disease that is transmissible to humans and that is currently incurable. BSE is caused by the prion protein (PrP), which adopts two conformers; PrPC is the native innocuous form, which is α-helix rich; and PrPSc is the β-sheet rich misfolded form, which is infectious and forms neurotoxic species. Acidic pH induces the conversion of PrPC to PrPSc. We have performed molecular dynamics simulations of bovine PrP at various pH regimes. An acidic pH environment induced conformational changes that were not observed in neutral pH simulations. Putative misfolded structures, with nonnative β-strands formed in the flexible N-terminal domain, were found in acidic pH simulations. Two distinct pathways were observed for the formation of nonnative β-strands: at low pH, hydrophobic contacts with M129 nucleated the nonnative β-strand; at mid-pH, polar contacts involving Q168 and D178 facilitated the formation of a hairpin at the flexible N-terminus. These mid- and low pH simulations capture the process of nonnative β-strand formation, thereby improving our understanding of how PrPC misfolds into the β-sheet rich PrPSc and how pH factors into the process. PMID:24970211

  19. Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH.

    PubMed

    Cheng, Chin Jung; Daggett, Valerie

    2014-01-01

    Bovine spongiform encephalopathy (BSE), or mad cow disease, is a fatal neurodegenerative disease that is transmissible to humans and that is currently incurable. BSE is caused by the prion protein (PrP), which adopts two conformers; PrPC is the native innocuous form, which is α-helix rich; and PrPSc is the β-sheet rich misfolded form, which is infectious and forms neurotoxic species. Acidic pH induces the conversion of PrPC to PrPSc. We have performed molecular dynamics simulations of bovine PrP at various pH regimes. An acidic pH environment induced conformational changes that were not observed in neutral pH simulations. Putative misfolded structures, with nonnative β-strands formed in the flexible N-terminal domain, were found in acidic pH simulations. Two distinct pathways were observed for the formation of nonnative β-strands: at low pH, hydrophobic contacts with M129 nucleated the nonnative β-strand; at mid-pH, polar contacts involving Q168 and D178 facilitated the formation of a hairpin at the flexible N-terminus. These mid- and low pH simulations capture the process of nonnative β-strand formation, thereby improving our understanding of how PrPC misfolds into the β-sheet rich PrPSc and how pH factors into the process. PMID:24970211

  20. Effect of pH on fecal recovery of energy derived from volatile fatty acids.

    PubMed

    Kien, C L; Liechty, E A

    1987-01-01

    We assessed the effect of pH on volatilization of short-chain fatty acids during lyophilization. Acetic, propionic, valeric, and butyric acids were added to a fecal homogenate in amounts sufficient to raise the energy density by 18-27%. Fecal homogenate samples were either acidified (pH 2.8-3.2), alkalinized (pH 7.9-8.7), or left unchanged (4.0-4.8) prior to lyophilization and subsequent bomb calorimetry. Alkalinizing the fecal samples prevented the 20% loss of energy derived from each of these volatile fatty acids observed in samples either acidified or without pH adjustment. These data suggest that in energy balance studies involving subjects with active colonic fermentation, fecal samples should be alkalinized prior to lyophilization and bomb calorimetry. PMID:3681570

  1. The absorption of acetylsalicylic acid from the stomach in relation to intragastric pH.

    PubMed

    Dotevall, G; Ekenved, G

    1976-01-01

    A comparative study on the effect of a buffered (pH 6.5) and an unbuffered (pH 2.9) solution of acetylsalicylic acid (ASA) on gastric pH, gastric emptying, and gastric absorption of ASA was performed in 10 healthy volunteers. Gastric pH was recorded using radiotelemetry. Gastric emptying and gastric absorption was studied with an aspiration technique and phenol red as nonabsorbable marker. Administration of the unbuffered solution to the fasting subjects resulted in a gastric pH of about 2 and absorption of ASA from the stomach was found to occur. The buffered solution of ASA increased gastric pH to above 5 and gastric absorption of ASA was found to be significantly less than after the unbuffered solution. The buffered solution was emptied from the stomach more rapidly than the unbuffered one. PMID:12558

  2. Effects of gaseous ammonia on intracellular pH values in leaves of C 3- and C 4-plants

    NASA Astrophysics Data System (ADS)

    Yin, Zu-Hua; Kaiser, Werner; Heber, Ulrich; Raven, John A.

    Responses of cytosolic and vacuolar pH to different concentrations (1.3-5.4 μmol NH 3 mol -1 gas or 0.940-3.825 mg NH 3 m -3 gas) of gaseous NH 3 were studied in experiments of 3 h duration by recording changes in fluorescence of pyranine and esculin in leaves of C 3 and C 4 plants. After a lag phase of 0.5-4 min, the uptake of NH 3 at 50-200 nmol m -2 leaf area s -1 increased pyranine fluorescence, indicating cytosolic alkalinization in leaves of Pelargonium zonale L. (C 3) and Amaranthus caudatus L. (C 4). A smaller increase in esculin fluorescence induced by NH 3 indicated some vacuolar alkalization in a Spinacia oleracea L. leaf. Photosynthesis and transpiration remained unchanged during exposure of illuminated leaves to NH 3 for up to 30 min (the maximum tested). CO 2 concentrations influenced the extent of cytosolic alkalinization. 500 μmol CO 2 mol -1 gas suppressed the NH 3-induced cytosolic alkalinization relative to that found in 16 μmol CO 2 mol -1 gas. The suppressing effect of CO 2 on NH 3-induced alkalization was larger in illuminated leaves of the C 4Amaranthus than the C 3Pelargonium. These results indicate that the alkaline pH shift caused by solution and protonation of NH 3 in aqueous leaf compartments is affected by assimilation of NH 3.

  3. Photoproduction of glyoxylic acid in model wine: Impact of sulfur dioxide, caffeic acid, pH and temperature.

    PubMed

    Grant-Preece, Paris; Schmidtke, Leigh M; Barril, Celia; Clark, Andrew C

    2017-01-15

    Glyoxylic acid is a tartaric acid degradation product formed in model wine solutions containing iron and its production is greatly increased by exposure to UV-visible light. In this study, the combined effect of sulfur dioxide, caffeic acid, pH and temperature on the light-induced (⩾300nm) production of glyoxylic acid in model wine containing tartaric acid and iron was investigated using a Box-Behnken experimental design and response surface methodology (RSM). Glyoxylic acid produced in the irradiated model wine was present in free and hydrogen sulfite adduct forms and the measured total, free and percentage free glyoxylic acid values were modeled using RSM. Sulfur dioxide significantly decreased the total amount of glyoxylic acid produced, but could not prevent its production, while caffeic acid showed no significant impact. The interaction between pH and temperature was significant, with low pH values and low temperatures giving rise to higher levels of total glyoxylic acid. PMID:27542478

  4. Extra- and Intracellular pH and Membrane Potential Changes Induced by K+, Cl−, H2PO4−, and NO3− Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum1

    PubMed Central

    Ullrich, Cornelia I.; Novacky, Anton J.

    1990-01-01

    Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl− and H2PO4− uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K+ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K+ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl− FC induced intracellular acidification. Since H+ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K+/A− symport or HCO3−/A− antiport for anion uptake. Though not suitable to reveal the actual H+/A− stoichiometry, the results are consistent with an H+/A− cotransport mechanism. PMID:16667890

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

    PubMed

    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

  6. Spherical Nucleic Acids as Intracellular Agents for Nucleic Acid Based Therapeutics

    NASA Astrophysics Data System (ADS)

    Hao, Liangliang

    Recent functional discoveries on the noncoding sequences of human genome and transcriptome could lead to revolutionary treatment modalities because the noncoding RNAs (ncRNAs) can be applied as therapeutic agents to manipulate disease-causing genes. To date few nucleic acid-based therapeutics have been translated into the clinic due to challenges in the delivery of the oligonucleotide agents in an effective, cell specific, and non-toxic fashion. Unmodified oligonucleotide agents are destroyed rapidly in biological fluids by enzymatic degradation and have difficulty crossing the plasma membrane without the aid of transfection reagents, which often cause inflammatory, cytotoxic, or immunogenic side effects. Spherical nucleic acids (SNAs), nanoparticles consisting of densely organized and highly oriented oligonucleotides, pose one possible solution to circumventing these problems in both the antisense and RNA interference (RNAi) pathways. The unique three dimensional architecture of SNAs protects the bioactive oligonucleotides from unspecific degradation during delivery and supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. Owing to their unique structure, SNAs are able to cross cell membranes and regulate target genes expression as a single entity, without triggering the cellular innate immune response. Herein, my thesis has focused on understanding the interactions between SNAs and cellular components and developing SNA-based nanostructures to improve therapeutic capabilities. Specifically, I developed a novel SNA-based, nanoscale agent for delivery of therapeutic oligonucleotides to manipulate microRNAs (miRNAs), the endogenous post-transcriptional gene regulators. I investigated the role of SNAs involving miRNAs in anti-cancer or anti-inflammation responses in cells and in in vivo murine disease models via systemic injection. Furthermore, I explored using different strategies to construct

  7. Acidic pH promotes oligomerization and membrane insertion of the BclXL apoptotic repressor.

    PubMed

    Bhat, Vikas; Kurouski, Dmitry; Olenick, Max B; McDonald, Caleb B; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

    2012-12-01

    Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues. PMID:22960132

  8. Chronic autophagy is a cellular adaptation to tumor acidic pH microenvironments.

    PubMed

    Wojtkowiak, Jonathan W; Rothberg, Jennifer M; Kumar, Virendra; Schramm, Karla J; Haller, Edward; Proemsey, Joshua B; Lloyd, Mark C; Sloane, Bonnie F; Gillies, Robert J

    2012-08-15

    Tumor cell survival relies upon adaptation to the acidic conditions of the tumor microenvironment. To investigate potential acidosis survival mechanisms, we examined the effect of low pH (6.7) on human breast carcinoma cells. Acute low pH exposure reduced proliferation rate, induced a G1 cell cycle arrest, and increased cytoplasmic vacuolization. Gene expression analysis revealed elevated levels of ATG5 and BNIP3 in acid-conditioned cells, suggesting cells exposed to low pH may utilize autophagy as a survival mechanism. In support of this hypothesis, we found that acute low pH stimulated autophagy as defined by an increase in LC3-positive punctate vesicles, double-membrane vacuoles, and decreased phosphorylation of AKT and ribosomal protein S6. Notably, cells exposed to low pH for approximately 3 months restored their proliferative capacity while maintaining the cytoplasmic vacuolated phenotype. Although autophagy is typically transient, elevated autophagy markers were maintained chronically in low pH conditioned cells as visualized by increased protein expression of LC3-II and double-membrane vacuoles. Furthermore, these cells exhibited elevated sensitivity to PI3K-class III inhibition by 3-methyladenine. In mouse tumors, LC3 expression was reduced by systemic treatment with sodium bicarbonate, which raises intratumoral pH. Taken together, these results argue that acidic conditions in the tumor microenvironment promote autophagy, and that chronic autophagy occurs as a survival adaptation in this setting. PMID:22719070

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

    PubMed Central

    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

  10. Zn2+ induces hyperpolarization by activation of a K+ channel and increases intracellular Ca2+ and pH in sea urchin spermatozoa

    PubMed Central

    Granados-González, Gisela; de De la Torre, Lucia García; Nishigaki, Takuya; Darszon, Alberto

    2014-01-01

    Zinc (Zn2+) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn2+ was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pHi). In this study we found that submicromolar concentrations of free Zn2+ change membrane potential (Em) and increase the concentration of intracellular Ca2+ ([Ca2+]i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn2+ response in sperm of this species mainly involves an Em hyperpolarization caused by K+ channel activation. The pharmacological profile of the Zn2+-induced hyperpolarization indicates that the cGMP-gated K+ selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn2+. Considering that Zn2+ also induces [Ca2+]i fluctuations, our observations suggest that Zn2+ activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn2+ in male gamete function. PMID:25092071

  11. Microbial degradation of isosaccharinic acid at high pH

    PubMed Central

    Bassil, Naji M; Bryan, Nicholas; Lloyd, Jonathan R

    2015-01-01

    Intermediate-level radioactive waste (ILW), which dominates the radioactive waste inventory in the United Kingdom on a volumetric basis, is proposed to be disposed of via a multibarrier deep geological disposal facility (GDF). ILW is a heterogeneous wasteform that contains substantial amounts of cellulosic material encased in concrete. Upon resaturation of the facility with groundwater, alkali conditions will dominate and will lead to the chemical degradation of cellulose, producing a substantial amount of organic co-contaminants, particularly isosaccharinic acid (ISA). ISA can form soluble complexes with radionuclides, thereby mobilising them and posing a potential threat to the surrounding environment or ‘far field'. Alkaliphilic microorganisms sampled from a legacy lime working site, which is an analogue for an ILW-GDF, were able to degrade ISA and couple this degradation to the reduction of electron acceptors that will dominate as the GDF progresses from an aerobic ‘open phase' through nitrate- and Fe(III)-reducing conditions post closure. Furthermore, pyrosequencing analyses showed that bacterial diversity declined as the reduction potential of the electron acceptor decreased and that more specialised organisms dominated under anaerobic conditions. These results imply that the microbial attenuation of ISA and comparable organic complexants, initially present or formed in situ, may play a role in reducing the mobility of radionuclides from an ILW-GDF, facilitating the reduction of undue pessimism in the long-term performance assessment of such facilities. PMID:25062127

  12. Effect of inhibitors of Na+/H+-exchange and gastric H+/K+ ATPase on cell volume, intracellular pH and migration of human polymorphonuclear leucocytes

    PubMed Central

    Ritter, M; Schratzberger, P; Rossmann, H; Wöll, E; Seiler, K; Seidler, U; Reinisch, N; Kähler, C M; Zwierzina, H; Lang, H J; Lang, F; Paulmichl, M; Wiedermann, C J

    1998-01-01

    Stimulation of chemotaxis of human polymorphonuclear leucocytes (PMNs) with the chemoattractive peptide fMLP (N-formyl-Met-Leu-Phe) is paralleled by profound morphological and metabolic alterations like changes of intracellular pH (pHi) and cell shape. The present study was performed to investigate the interrelation of cell volume (CV) regulatory ion transport, pHi and migration of fMLP stimulated PMNs.Addition of fMLP to PMNs stimulated directed migration in Boyden chamber assays and was accompanied by rapid initial intracellular acidification and cell swelling.Inhibition of the Na+/H+ exchanger suppressed fMLP stimulated cell migration, accelerated the intracellular acidification and inhibited the fMLP-induced cell swelling.Step omission of extracellular Na+ caused intracellular acidification, which was accelerated by subsequent addition of gastric H+/K+ ATPase inhibitor SCH 28080, or by omission of extracellular K+ ions. In addition Na+ removal caused cell swelling, which was further enhanced by fMLP.H+/K+ATPase inhibitors omeprazole and SCH 28080 inhibited stimulated migration and blunted the fMLP-induced increase in CV.Increasing extracellular osmolarity by addition of mannitol to the extracellular solution caused cell shrinkage followed by regulatory volume increase, partially due to activation of the Na+/H+ exchanger. In fMLP-stimulated cells the CV increase was counteracted by simultaneous addition of mannitol. Under these conditions the fMLP stimulated migration was inhibited.The antibacterial activity of PMNs was not modified by Hoe 694 or omeprazole.Western analysis with a monoclonal anti gastric H+/K+ATPase β-subunit antibody detected a glycosylated 35 kD core protein in lysates of mouse and human gastric mucosa as well as in human PMNs.The results indicate that fMLP leads to cell swelling of PMNs due to activation of the Na+/H+ exchanger and a K+-dependent H+-extruding mechanism, presumably an H+/K+ ATPase. Inhibition of these ion transporters

  13. Initial pH of medium affects organic acids production but do not affect phosphate solubilization

    PubMed Central

    Marra, Leandro M.; de Oliveira-Longatti, Silvia M.; Soares, Cláudio R.F.S.; de Lima, José M.; Olivares, Fabio L.; Moreira, Fatima M.S.

    2015-01-01

    The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization. PMID:26273251

  14. Initial pH of medium affects organic acids production but do not affect phosphate solubilization.

    PubMed

    Marra, Leandro M; de Oliveira-Longatti, Silvia M; Soares, Cláudio R F S; de Lima, José M; Olivares, Fabio L; Moreira, Fatima M S

    2015-06-01

    The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization. PMID:26273251

  15. Calculation of downhole pH and delta pH in the presence of CO{sub 2} and organic acids

    SciTech Connect

    Garber, J.D.; Perkins, R.S.; Jangama, V.R.; Alapati, R.R.

    1996-08-01

    Acetic and formic acids have been found in the separator water of gas condensate wells containing CO{sub 2} and they are titrated as alkalinity. Traditional pH equations which neglect these acids and calculate pH based on alkalinity greatly over-predict the downhole pH. Since all scale calculations depend on an accurate pH value, a more sophisticated method of calculation has been developed. The methodology can be used to calculate the in-situ bulk pH and the saturation pH at different depths within a well. The difference in the saturation pH and the bulk pH is the delta pH a negative delta pH indicates a potential to scale whereas a positive value indicates a potential to corrode. The saturation pH is discussed with respect to iron carbonate saturation, but can be used for any other scale by making the appropriate changes.

  16. Relationship of Cell Sap pH to Organic Acid Change During Ion Uptake 1

    PubMed Central

    Hiatt, A. J.

    1967-01-01

    Excised roots of barley (Hordeum vulgare, var. Campana) were incubated in KCl, K2SO4, CaCl2, and NaCl solutions at concentrations of 10−5 to 10−2 n. Changes in substrate solution pH, cell sap pH, and organic acid content of the roots were related to differences in cation and anion absorption. The pH of expressed sap of roots increased when cations were absorbed in excess of anions and decreased when anions were absorbed in excess of cations. The pH of the cell sap shifted in response to imbalances in cation and anion uptake in salt solutions as dilute as 10−5 n. Changes in cell sap pH were detectable within 15 minutes after the roots were placed in 10−3 n K2SO4. Organic acid changes in the roots were proportional to expressed sap pH changes induced by unbalanced ion uptake. Changes in organic acid content in response to differential cation and anion uptake appear to be associated with the low-salt component of ion uptake. PMID:16656506

  17. Influence of isolation media on synaptosomal properties: Intracellular pH, pCa, and Ca sup 2+ uptake

    SciTech Connect

    Bandeira-Duarte, C.; Carvalho, C.A.; Cragoe Junior, E.J.; Carvalho, A.P. )

    1990-03-01

    Preparations of synaptosomes isolated in sucrose or in Na(+)-rich media were compared with respect to internal pH (pHi), internal Ca{sup 2+} concentration ((Ca{sup 2+})i), membrane potential and {sup 45}Ca{sup 2+} uptake due to K+ depolarization and Na{sup +}/Ca{sup 2+} exchange. We found that synaptosomes isolated in sucrose media have a pHi of 6.77 +/- 0.04 and a (Ca{sup 2+})i of about 260 nM, whereas synaptosomes isolated in Na(+)-rich ionic media have a pHi of 6.96 +/- 0.07 and a (Ca{sup 2+})i of 463 nM, but both types of preparations have similar membrane potentials of about -50 mV when placed in choline media. The sucrose preparation takes up Ca{sup 2+} only by voltage sensitive calcium channels (VSCC'S) when K(+)-depolarized, while the Na(+)-rich synaptosomes take up {sup 45}Ca{sup 2+} both by VSCC'S and by Na{sup +}/Ca{sup 2+} exchange. The amiloride derivative 2',4'-dimethylbenzamil (DMB), at 30 microM, inhibits both mechanisms of Ca{sup 2+} influx, but 5-(N-4-chlorobenzyl)-2',4' dimethylbenzamil (CBZ-DMB), at 30 microM, inhibits the Ca{sup 2+} uptake by VSCC'S, but not by Na+/Ca2+ exchange. Thus, DMB and CBZ-DMB permit distinguishing between Ca{sup 2+} flux through channels and through Na{sup +}/Ca{sup 2+} exchange. We point out that the different properties of the two types of synaptosomes studied account for some of the discrepancies in results reported in the literature for studies of Ca{sup 2+} fluxes and neurotransmitter release by different types of preparations of synaptosomes.

  18. 31P and 1H MRS of DB-1 Melanoma Xenografts: Lonidamine Selectively Decreases Tumor Intracellular pH and Energy Status and Sensitizes Tumors to Melphalan

    PubMed Central

    Nath, Kavindra; Nelson, David S.; Ho, Andrew; Lee, Seung-Cheol; Darpolor, Moses M.; Pickup, Stephen; Zhou, Rong; Heitjan, Daniel F.; Leeper, Dennis B.; Glickson, Jerry D.

    2012-01-01

    In vivo 31P MRS demonstrates that human melanoma xenografts in immunosuppressed mice treated with lonidamine (LND, 100 mg/kg, i.p.) exhibit a decrease in intracellular pH (pHi) from 6.90 ± 0.05 to 6.33 ± 0.10 (p < 0.001), a slight decrease in extracellular pH (pHe) from 7.00 ± 0.04 to 6.80 ± 0.07 (p > 0.05), and a monotonic decline in bioenergetics (NTP/Pi) by 66.8 ± 5.7% (p < 0.001) relative to the baseline level. Both bioenergetics and pHi decreases were sustained for at least 3 hr following LND treatment. Liver exhibited a transient intracellular acidification by 0.2 ± 0.1 pH units (p > 0.05) at 20 min post-LND with no significant change in pHe and a small transient decrease in bioenergetics, 32.9 ± 10.6 % (p > 0.05), at 40 min post-LND. No changes in pHi or ATP/Pi were detected in the brain (pHi, bioenergetics; p > 0.1) or skeletal muscle (pHi, pHe, bioenergetics; p > 0.1) for at least 120 min post-LND. Steady-state tumor lactate monitored by 1H MRS with a selective multiquantum pulse sequence with Hadamard localization increased ~3-fold (p = 0.009). Treatment with LND increased systemic melanoma response to melphalan (LPAM; 7.5 mg/kg, i.v.) producing a growth delay of 19.9 ± 2.0 d (tumor doubling time = 6.15 ± 0.31d, log10 cell-kill = 0.975 ± 0.110, cell-kill = 89.4 ± 2.2%) compared to LND alone of 1.1 ± 0.1 d and LPAM alone of 4.0 ± 0.0 d. The study demonstrates that the effects of LND on tumor pHi and bioenergetics may sensitize melanoma to pH-dependent therapeutics such as chemotherapy with alkylating agents or hyperthermia. PMID:22745015

  19. Influence of Acidic pH on Hydrogen and Acetate Production by an Electrosynthetic Microbiome

    PubMed Central

    LaBelle, Edward V.; Marshall, Christopher W.; Gilbert, Jack A.; May, Harold D.

    2014-01-01

    Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm2 at −765 mV (0.065 mA/cm2 sterile control at −800 mV) by the Acetobacterium-dominated community. Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m3/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m3/day formate, and 3.1 kg/m3/day acetate ( = 4.7 kg CO2 captured). PMID:25333313

  20. Transcriptome Profiling of Shewanella oneidensis Gene Expressionfollowing Exposure to Acidic and Alkaline pH

    SciTech Connect

    Leaphart, Adam B.; Thompson, Dorothea K.; Huang, Katherine; Alm,Eric; Wan, Xiu-Feng; Arkin, Adam P.; Brown, Steven D.; Wu, Liyou; Yan,Tingfen; Liu, Xueduan; Wickham, Gene S.; Zhou, Jizhong

    2007-04-02

    The molecular response of Shewanella oneidensis MR-1 tovariations in extracellular pH was investigated based on genomewide geneexpression profiling. Microarray analysis revealed that cells elicitedboth general and specific transcriptome responses when challenged withenvironmental acid (pH 4) or base (pH 10) conditions over a 60-minperiod. Global responses included the differential expression of genesfunctionally linked to amino acid metabolism, transcriptional regulationand signal transduction, transport, cell membrane structure, andoxidative stress protection. Response to acid stress included theelevated expression of genes encoding glycogen biosynthetic enzymes,phosphate transporters, and the RNA polymerase sigma-38 factor (rpoS),whereas the molecular response to alkaline pH was characterized byupregulation of nhaA and nhaR, which are predicted to encode an Na+/H+antiporter and transcriptional activator, respectively, as well assulfate transport and sulfur metabolism genes. Collectively, theseresults suggest that S. oneidensis modulates multiple transporters, cellenvelope components, and pathways of amino acid consumption and centralintermediary metabolism as part of its transcriptome response to changingexternal pH conditions.

  1. Influence of acidic pH on hydrogen and acetate production by an electrosynthetic microbiome

    SciTech Connect

    LaBelle, Edward V.; Marshall, Christopher W.; Gilbert, Jack A.; May, Harold D.; Battista, John R.

    2014-10-15

    Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (~5). Hydrogen production by biocathodes poised at -600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ~5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ~6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm2 at -765 mV (0.065 mA/cm2 sterile control at -800 mV) by the Acetobacterium-dominated community. Supplying -800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m3/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m3/day formate, and 3.1 kg/m3/day acetate ( = 4.7 kg CO2 captured).

  2. Influence of acidic pH on hydrogen and acetate production by an electrosynthetic microbiome

    DOE PAGESBeta

    LaBelle, Edward V.; Marshall, Christopher W.; Gilbert, Jack A.; May, Harold D.; Battista, John R.

    2014-10-15

    Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (~5). Hydrogen production by biocathodes poised at -600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ~5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ~6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm2 at -765 mV (0.065 mA/cm2 sterile control at -800 mV) by the Acetobacterium-dominatedmore » community. Supplying -800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m3/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m3/day formate, and 3.1 kg/m3/day acetate ( = 4.7 kg CO2 captured).« less

  3. Influence of organic acids on the pH and acid-neutralizing capacity of Adirondack Lakes

    NASA Astrophysics Data System (ADS)

    Munson, R. K.; Gherini, S. A.

    1993-04-01

    Past approaches for evaluating the effects of organic acids on the acid-base characteristics of surface waters have typically treated them solely as weak acids. Analysis of data collected by the Adirondack Lakes Survey Corporation (ALSC) from 1469 lakes throughout the Adirondack region shows that this approach is not valid. While the data indicate that natural organics contain a continuum of acid functional groups, many of which display weak acid characteristics, a significant fraction of the organic acid is strong (pKa < 3). Dissolved organic carbon (DOC) contributes 4.5-5 μeq/mg DOC of strong acid to solution. The associated anions make a negative contribution to Gran acid-neutralizing capacity (ANC). Because organic anions can produce negative Gran ANC values, the common practice of considering negative values of Gran ANC evidence of acidification solely by mineral acids is not valid. The strength of organic acids also influences the observed deviation between Gran ANC values and ANC values calculated as the difference between base cation and mineral acid anion concentrations (CB - CA). Ninety percent of the deviation is due to the presence of strong organics while the remaining 10% is due to DOC-induced curvature in the F1 Gran function. Organic acids can also strongly influence pH. Their largest effects were found in the 0-50 μeq/L Gran ANC range where they depressed pH by up to 1.5 units. In addition, a method for predicting changes in pH in response to changes in mineral acidity, DOC, or both without having to rely on inferred thermodynamic constants and the uncertainties associated with them has been developed. Using the predictive method, the response of representative lakes from four sensitive lake classes to a 15-μeq/L decrease in mineral acidity ranged from +0.17 to +0.38 pH units. If concurrent increases in DOC are considered, the pH changes would be even smaller.

  4. A novel glyceryl monoolein-bearing cubosomes for gambogenic acid: Preparation, cytotoxicity and intracellular uptake.

    PubMed

    Luo, Qing; Lin, Tongyuan; Zhang, Cai Yuan; Zhu, Tingting; Wang, Lei; Ji, Zhaojie; Jia, Buyun; Ge, Tao; Peng, Daiyin; Chen, Weidong

    2015-09-30

    Lyotropic cubic liquid crystalline nanoparticles, also known as 'cubosomes', have been tested as effective carriers for a variety of drugs due to their ability to enhance delivery efficiency and reduced drug side effects. Cubosomes are colloidal carriers composed of biodegradable Glyceryl monooleate and F127. Being composed of well tolerable and physiological materials, these carriers are well tolerated, compatible and non-toxic. In this study, therefore, we developed a novel, water-soluble, glyceryl monooleate and F127 based multiblock copolymer for Gambogenic acid (GNA) by emulsion-evaporation and low temperature-solidification technique. Physicochemical properties, in vitro cytotoxicity, cellular uptake and in vivo pharmacokinetic of GNA-loaded cubosomes (GNA-Cubs) were investigated. The results revealed that GNA-Cubs were spherical or ellipsoidal monocellular by dynamic light scattering, meanwhile, 150-250nm in mean size with narrow polydispersity indexas determined by transmission electron microscopy. Small angle X-ray scattering indicated that GNA-Cubs retain the Pn3m cubic symmetry. Compared with GNA solution, GNA-Cubs exhibited markedly prolonged inhibitory activity in SMMC-7721 cells, as well as time-dependent increases in intra-cellular uptake. Furthermore, in vivo pharmacokinetic study showed that the Cmax values and the AUC of GNA-Cubs were higher than GNA solution approximately 1.2-fold and 9.1-fold, respectively. In conclusion, the results showed that the cubic liquid crystalline nanoparticles could be a potentially nanocarrier in the delivery of GNA for cancer therapy. PMID:26209071

  5. Bacteriophage SP82G Inhibition of an Intracellular Deoxyribonucleic Acid Inactivation Process in Bacillus subtilis1

    PubMed Central

    McAllister, William T.; Green, D. MacDonald

    1972-01-01

    The stability of SP82G bacteriophage deoxyribonucleic acid (DNA) after its uptake by competent Bacillus subtilis was examined by determining the ability of superinfecting phage particles to rescue genetic markers carried by the infective DNA. These experiments show that a DNA inactivation process within the cell is inhibited after infection of the cell by intact phage particles. The inhibition is maximally expressed 6 min after phage infection and is completely prevented by the addition of chloramphenicol at the time of infection. The protective effect of this function extends even to infective DNA which was present in the cell before the addition of intact phage. Continued protein synthesis does not appear to be a requirement for the maintenance of the inhibition. In an analogous situation, if infectious centers resulting from singly infecting phage particles are exposed to chloramphenicol shortly after the time of infection, an exponential decrease in the survival of infectious centers with time held in chloramphenicol is observed. If the addition of chloramphenicol is delayed until 6 min after infection, the infectious centers are resistant to chloramphenicol. The sensitivity of infectious centers treated with chloramphenicol at early times after infection is strongly dependent upon the multiplicity of infection and is consistent with a model of multiplicity reactivation. These results indicate that injected DNA is also susceptible to the intracellular inactivation process and suggest that the inhibition of this system is necessary for the successful establishment of an infectious center. PMID:4625174

  6. Target-specific intracellular delivery of siRNA using degradable hyaluronic acid nanogels.

    PubMed

    Lee, Hyukjin; Mok, Hyejung; Lee, Soohyeon; Oh, Yu-Kyoung; Park, Tae Gwan

    2007-06-01

    Novel hyaluronic acid (HA) nanogels physically encapsulating small interfering RNA (siRNA) were fabricated by an inverse water-in-oil emulsion method. Thiol-conjugated HA dissolved in aqueous emulsion droplets was ultrasonically crosslinked via the formation of disulfide linkages to produce HA nanogels with a size distribution from 200 to 500 nm. Green fluorescence protein (GFP) siRNA was physically entrapped within the HA nanogels during the emulsion/crosslinking process. The HA/siRNA nanogels were readily taken up by HA receptor positive cells (HCT-116 cells) having HA-specific CD44 receptors on the surface. Release rates of siRNA from the HA nanogels could be modulated by changing the concentration of glutathione (GSH) in the buffer solution, indicating that the degradation/erosion of disulfide crosslinked HA nanogels, triggered by an intracellular reductive agent, controlled the release pattern of siRNA. When HA nanogels containing GFP siRNA were co-transfected with GFP plasmid/Lipofectamine to HCT-116 cells, a significant extent of GFP gene silencing was observed in both serum and non-serum conditions. The gene silencing effect was reduced in the presence of free HA in the transfection medium, revealing that HA nanogels were selectively taken up by HCT-116 cells via receptor mediated endocytosis. PMID:17408798

  7. Effects of saliva on starch-thickened drinks with acidic and neutral pH.

    PubMed

    Hanson, Ben; Cox, Ben; Kaliviotis, Efstathios; Smith, Christina H

    2012-09-01

    Powdered maize starch thickeners are used to modify drink consistency in the clinical management of dysphagia. Amylase is a digestive enzyme found in saliva which breaks down starch. This action is dependent on pH, which varies in practice depending on the particular drink. This study measured the effects of human saliva on the viscosity of drinks thickened with a widely used starch-based thickener. Experiments simulated a possible clinical scenario whereby saliva enters a cup and contaminates a drink. Citric acid (E330) was added to water to produce a controlled range of pH from 3.0 to 7.0, and several commercially available drinks with naturally low pH were investigated. When saliva was added to thickened water, viscosity was reduced to less than 1% of its original value after 10-15 min. However, lowering pH systematically slowed the reduction in viscosity attributable to saliva. At pH 3.5 and below, saliva was found to have no significant effect on viscosity. The pH of drinks in this study ranged from 2.6 for Coca Cola to 6.2 for black coffee. Again, low pH slowed the effect of saliva. For many popular drinks, having pH of 3.6 or less, viscosity was not significantly affected by the addition of saliva. PMID:22210234

  8. Development of Online Spectroscopic pH Monitoring for Nuclear Fuel Reprocessing Plants: Weak Acid Schemes.

    PubMed

    Casella, Amanda J; Ahlers, Laura R H; Campbell, Emily L; Levitskaia, Tatiana G; Peterson, James M; Smith, Frances N; Bryan, Samuel A

    2015-05-19

    In nuclear fuel reprocessing, separating trivalent minor actinides and lanthanide fission products is extremely challenging and often necessitates tight pH control in TALSPEAK (Trivalent Actinide-Lanthanide Separation by Phosphorus reagent Extraction from Aqueous Komplexes) separations. In TALSPEAK and similar advanced processes, aqueous pH is one of the most important factors governing the partitioning of lanthanides and actinides between an aqueous phase containing a polyaminopolycarboxylate complexing agent and a weak carboxylic acid buffer and an organic phase containing an acidic organophosphorus extractant. Real-time pH monitoring would significantly increase confidence in the separation performance. Our research is focused on developing a general method for online determination of the pH of aqueous solutions through chemometric analysis of Raman spectra. Spectroscopic process-monitoring capabilities, incorporated in a counter-current centrifugal contactor bank, provide a pathway for online, real-time measurement of solution pH. The spectroscopic techniques are process-friendly and can be easily configured for online applications, whereas classic potentiometric pH measurements require frequent calibration/maintenance and have poor long-term stability in aggressive chemical and radiation environments. Raman spectroscopy discriminates between the protonated and deprotonated forms of the carboxylic acid buffer, and the chemometric processing of the Raman spectral data with PLS (partial least-squares) regression provides a means to quantify their respective abundances and therefore determine the solution pH. Interpretive quantitative models have been developed and validated under a range of chemical composition and pH conditions using a lactic acid/lactate buffer system. The developed model was applied to new spectra obtained from online spectral measurements during a solvent extraction experiment using a counter-current centrifugal contactor bank. The model

  9. Oesophageal clearance of acid and bile: a combined radionuclide, pH, and Bilitec study

    PubMed Central

    Koek, G H; Vos, R; Flamen, P; Sifrim, D; Lammert, F; Vanbilloen, B; Janssens, J; Tack, J

    2004-01-01

    Background: Studies combining pH and Bilitec monitoring found a high prevalence of both acid and duodeno-gastro-oesophageal reflux in severe reflux disease. Clearance of refluxed material is a major defence mechanism against reflux. Several studies have been devoted to oesophageal acid clearance but oesophageal clearance of refluxed duodenal contents (DC) has rarely been addressed. Aim: To compare oesophageal acid and DC clearance. Methods: Ten healthy volunteers (five women, mean age 23 (1) years) were studied. Firstly, a balloon tip catheter, positioned in the duodenum under fluoroscopy, was used to aspirate DC after stimulation by a high caloric liquid meal (200 ml, 300 kcal). During the second session, pH and Bilitec probes were positioned 5 cm above the lower oesophageal sphincter and a small infusion catheter was introduced into the proximal oesophagus. The subject was placed supine under a gamma camera. One of two different solutions (DC mixed with 0.2 mCi Tc99m pertechnetate or citric acid (pH 2) mixed with 0.2 mCi Tc99m pertechnetate) was infused into the proximal oesophagus and the subject was instructed to swallow at 20 second intervals. Clearance was assessed using scintigraphy (dynamic acquisition, one frame per second in the anterior view; calculation of time to clear peak counts to background level), pH (time to pH<4) or Bilitec (time absorbance >0.14) monitoring, with or without continuous saliva aspiration. Each condition was studied twice in a randomised design; measurement time was four minutes, interrupted by water flushing, with a two minute rest period. Results are given as mean (SEM) and were compared by Student’s t test and Pearson correlation. Results: Scintigraphic evaluation showed a volume clearance time of 29 (3) seconds for acid and 28 (9) seconds for DC (NS). Saliva aspiration had no significant influence on volume clearance of acid or DC (28 (4) and 30 (13) seconds, respectively; NS). pH monitoring showed an acid clearance time of

  10. Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH.

    PubMed

    Sriranganadane, Dev; Waridel, Patrice; Salamin, Karine; Feuermann, Marc; Mignon, Bernard; Staib, Peter; Neuhaus, Jean-Marc; Quadroni, Manfredo; Monod, Michel

    2011-11-01

    The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi. PMID:21919205

  11. Effect of inhibitors of arachidonic acid metabolism on efflux of intracellular enzymes from skeletal muscle following experimental damage.

    PubMed Central

    Jackson, M J; Wagenmakers, A J; Edwards, R H

    1987-01-01

    The role of arachidonic acid metabolism in the efflux of intracellular enzymes from damaged skeletal muscle has been examined in vitro using inhibitors of cyclo-oxygenase and lipoxygenase enzymes. Damage to skeletal muscle induced by either calcium ionophore A23187 (25 microM) or dinitrophenol (1 mM) caused an increase in the efflux of prostaglandins E2 and F2 alpha together with a large efflux of intracellular creatine kinase. Use of a cyclo-oxygenase inhibitor completely prevented the efflux of prostaglandins, but had no effect on creatine kinase efflux. However, several agents having the ability to inhibit lipoxygenase enzymes dramatically reduced creatine kinase efflux following damage. These data suggest that a product or products of lipoxygenase enzymes may be mediators of the changes in plasma membrane integrity which permit efflux of intracellular enzymes as a consequence of skeletal muscle damage. PMID:3109374

  12. Candida albicans erythroascorbate peroxidase regulates intracellular methylglyoxal and reactive oxygen species independently of D-erythroascorbic acid.

    PubMed

    Kwak, Min-Kyu; Song, Sung-Hyun; Ku, MyungHee; Kang, Sa-Ouk

    2015-07-01

    Candida albicans D-erythroascorbate peroxidase (EAPX1), which can catalyze the oxidation of D-erythroascorbic acid (EASC) to water, was observed to be inducible in EAPX1-deficient and EAPX1-overexpressing cells via activity staining. EAPX1-deficient cells have remarkably increased intracellular reactive oxygen species and methylglyoxal independent of the intracellular EASC content. The increased methylglyoxal caused EAPX1-deficient cells to activate catalase-peroxidase and cytochrome c peroxidase, which led to defects in cell growth, viability, mitochondrial respiration, filamentation and virulence. These findings indicate that EAPX1 mediates cell differentiation and virulence by regulating intracellular methylglyoxal along with oxidative stresses, regardless of endogenous EASC biosynthesis or alternative oxidase expression. PMID:25957768

  13. Optimization of pH values to formulate the bireagent kit for serum uric acid assay.

    PubMed

    Huang, Ya; Chen, Yuanxiang; Yang, Xiaolan; Zhao, Hua; Hu, Xiaolei; Pu, Jun; Liao, Juan; Long, Gaobo; Liao, Fei

    2015-01-01

    A new formulation of the bireagent kit for serum uric acid assay was developed based on the effects of pH on enzyme stability. At 4 °C, half-lives of uricases from Bacillus fastidious and Arthrobacter globiforms were longer than 15 months at pH 9.2, but became shorter at pH below 8.0; half-lives of ascorbate oxidase and peroxidase were comparable at pH 6.5 and 7.0, but became much shorter at pH higher than 7.4. In the new formulation of the bireagent kit, Reagent A contained peroxidase, 4-aminoantipyrine, and ascorbate oxidase in 50 mM phosphate buffer at pH 6.5; Reagent B contained B. fastidious or A. globiforms uricase in 50 mM sodium borate buffer at pH 9.2; Reagents A and B were mixed at 4:1 to produce a final pH from 7.2 to 7.6 for developing a stable color. The new bireagent kit consumed smaller quantities of three enzymes for the same shelf life. With the new bireagent kit, there were linear responses of absorbance at 546 nm to uric acid up to 34 mM in reaction mixtures and a good correlation of uric acid levels in clinical sera with those by a commercial kit, but stronger resistance to ascorbate. Therefore, the new formulation was advantageous. PMID:24673428

  14. Changes in soil pH across England and Wales in response to decreased acid deposition

    NASA Astrophysics Data System (ADS)

    Kirk, G. J. D.; Bellamy, P. H.

    2009-04-01

    In our recent analysis of data from the National Soil Inventory of England and Wales, we found widespread changes in soil pH across both countries between the two samplings of the Inventory. In general, soil pH increased - i.e. soils became less acid - under all land uses. The Inventory was first sampled in 1978-83 on a 5-km grid over the whole area. This yielded about 6,000 sites of which 5,662 could be sampled for soil. Roughly 40% of the sites were re-sampled at intervals from 12 to 25 years after the original sampling - in 1994/96 for agricultural land and in 2002/03 for non-agricultural. Exactly the same sampling and analytical protocols were used in the two samplings. In arable soils, the increase in pH was right across the range, whereas in grassland soils the main increase was at the acid end of the scale (pH < 5.5) with a small increase above pH 7. Some part of the change is likely to have been due to changes in land management. This includes better targeting of agricultural lime on acid soils; changes in nitrogen fertilizer use; deeper ploughing bringing up more calcareous subsoil on soils on calcareous materials; and so forth. However a major driver appears to have been decreased acid deposition to land. The total amounts of nitrogen compounds deposited were relatively unchanged over the survey period, but the amounts of acidifying sulphur compounds decreased by approximately 50%. We constructed a linear regression model to assess the relation between the rate of change in pH (normalised to an annual basis) and the rate of change in acid deposition, as modified by soil properties (pH, clay content, organic matter content), rainfall and past acid deposition. We used data on rainfall and acid deposition over the survey period on the same 5-km grid as the NSI data. We fitted the model separately for each land use category. The results for arable land showed a significant effect of the change in rate of acid deposition, though a significant part of the

  15. Metal Interactions with Microbial Biofilms in Acidic and Neutral pH Environments

    PubMed Central

    Ferris, F. G.; Schultze, S.; Witten, T. C.; Fyfe, W. S.; Beveridge, T. J.

    1989-01-01

    Microbial biofilms were grown on strips of epoxy-impregnated filter paper submerged at four sites in water contaminated with metals from mine wastes. At two sample stations, the water was acidic (pH 3.1); the other sites were in a lake restored to a near neutral pH level by application of a crushed limestone slurry. During a 17-week study period, planktonic bacterial counts increased from 101 to 103 CFU/ml at all sites. Biofilm counts increased rapidly over the first 5 weeks and then leveled to 104 CFU/cm2 in the neutral pH system and 103 CFU/cm2 at the acidic sites. In each case, the biofilms bound Mn, Fe, Ni, and Cu in excess of the amounts adsorbed by control strips covered with nylon filters (pore size, 0.22 μm) to exclude microbial growth; Co bound under neutral conditions but not under acidic conditions. Conditional adsorption capacity constants, obtained graphically from the data, showed that biofilm metal uptake at a neutral pH level was enhanced by up to 12 orders of magnitude over acidic conditions. Similarly, adsorption strength values were usually higher at elevated pH levels. In thin sections of the biofilms, encapsulated bacterial cells were commonly found enmeshed together in microcolonies. The extracellular polymers often contained iron oxide precipitates which generated weak electron diffraction patterns with characteristic reflections for ferrihydrite (Fe2O3 · H2O) at d equaling 0.15 and 0.25 nm. At neutral pH levels, these deposits incorporated trace amounts of Si and exhibited a granular morphology, whereas acicular crystalloids containing S developed under acidic conditions. Images PMID:16347914

  16. A new boronic acid fluorescent sensor based on fluorene for monosaccharides at physiological pH

    NASA Astrophysics Data System (ADS)

    Hosseinzadeh, Rahman; Mohadjerani, Maryam; Pooryousef, Mona; Eslami, Abbas; Emami, Saeed

    2015-06-01

    Fluorescent boronic acids are very useful fluorescent sensor for detection of biologically important saccharides. Herein we synthesized a new fluorene-based fluorescent boronic acid that shows significant fluorescence changes upon addition of saccharides at physiological pH. Upon addition of fructose, sorbitol, glucose, galactose, ribose, and maltose at different concentration to the solution of 7-(dimethylamino)-9,9-dimethyl-9H-fluoren-2-yl-2-boronic acid (7-DMAFBA, 1), significant decreases in fluorescent intensity were observed. It was found that this boronic acid has high affinity (Ka = 3582.88 M-1) and selectivity for fructose over glucose at pH = 7.4. The sensor 1 showed a linear response toward D-fructose in the concentrations ranging from 2.5 × 10-5 to 4 × 10-4 mol L-1 with the detection limit of 1.3 × 10-5 mol L-1.

  17. Toxicity and intracellular accumulation of bile acids in sandwich-cultured rat hepatocytes: role of glycine conjugates.

    PubMed

    Chatterjee, Sagnik; Bijsmans, Ingrid T G W; van Mil, Saskia W C; Augustijns, Patrick; Annaert, Pieter

    2014-03-01

    Excessive intrahepatic accumulation of bile acids (BAs) is a key mechanism underlying cholestasis. The aim of this study was to quantitatively explore the relationship between cytotoxicity of BAs and their intracellular accumulation in sandwich-cultured rat hepatocytes (SCRH). Following exposure of SCRH (on day-1 after seeding) to various BAs for 24h, glycine-conjugated BAs were most potent in exerting toxicity. Moreover, unconjugated BAs showed significantly higher toxicity in day-1 compared to day-3 SCRH. When day-1/-3 SCRH were exposed (0.5-4h) to 5-100μM (C)DCA, intracellular levels of unconjugated (C)DCA were similar, while intracellular levels of glycine conjugates were up to 4-fold lower in day-3 compared to day-1 SCRH. Sinusoidal efflux was by far the predominant efflux pathway of conjugated BAs both in day-1 and day-3 SCRH, while canalicular BA efflux showed substantial interbatch variability. After 4h exposure to (C)DCA, intracellular glycine conjugate levels were at least 10-fold higher than taurine conjugate levels. Taken together, reduced BA conjugate formation in day-3 SCRH results in lower intracellular glycine conjugate concentrations, explaining decreased toxicity of (C)DCA in day-3 versus day-1 SCRH. Our data provide for the first time a direct link between BA toxicity and glycine conjugate exposure in SCRH. PMID:24211540

  18. pH-responsive biocompatible fluorescent polymer nanoparticles based on phenylboronic acid for intracellular imaging and drug delivery

    NASA Astrophysics Data System (ADS)

    Li, Shengliang; Hu, Kelei; Cao, Weipeng; Sun, Yun; Sheng, Wang; Li, Feng; Wu, Yan; Liang, Xing-Jie

    2014-10-01

    To address current medical challenges, there is an urgent need to develop drug delivery systems with multiple functions, such as simultaneous stimuli-responsive drug release and real-time imaging. Biocompatible polymers have great potential for constructing smart multifunctional drug-delivery systems through grafting with other functional ligands. More importantly, novel biocompatible polymers with intrinsic fluorescence emission can work as theranostic nanomedicines for real-time imaging and drug delivery. Herein, we developed a highly fluorescent nanoparticle based on a phenylboronic acid-modified poly(lactic acid)-poly(ethyleneimine)(PLA-PEI) copolymer loaded with doxorubicin (Dox) for intracellular imaging and pH-responsive drug delivery. The nanoparticles exhibited superior fluorescence properties, such as fluorescence stability, no blinking and excitation-dependent fluorescence behavior. The Dox-loaded fluorescent nanoparticles showed pH-responsive drug release and were more effective in suppressing the proliferation of MCF-7 cells. In addition, the biocompatible fluorescent nanoparticles could be used as a tool for intracellular imaging and drug delivery, and the process of endosomal escape was traced by real-time imaging. These pH-responsive and biocompatible fluorescent polymer nanoparticles, based on phenylboronic acid, are promising tools for intracellular imaging and drug delivery.To address current medical challenges, there is an urgent need to develop drug delivery systems with multiple functions, such as simultaneous stimuli-responsive drug release and real-time imaging. Biocompatible polymers have great potential for constructing smart multifunctional drug-delivery systems through grafting with other functional ligands. More importantly, novel biocompatible polymers with intrinsic fluorescence emission can work as theranostic nanomedicines for real-time imaging and drug delivery. Herein, we developed a highly fluorescent nanoparticle based on a

  19. Dissolved Divalent Metal and pH Effects on Amino Acid Polymerization: A Thermodynamic Evaluation

    NASA Astrophysics Data System (ADS)

    Kitadai, Norio

    2016-06-01

    Polymerization of amino acids is a fundamentally important step for the chemical evolution of life. Nevertheless, its response to changing environmental conditions has not yet been well understood because of the lack of reliable quantitative information. For thermodynamics, detailed prediction over diverse combinations of temperature and pH has been made only for a few amino acid-peptide systems. This study used recently reported thermodynamic dataset for the polymerization of the simplest amino acid "glycine (Gly)" to its short peptides (di-glycine and tri-glycine) to examine chemical and structural characteristics of amino acids and peptides that control the temperature and pH dependence of polymerization. Results showed that the dependency is strongly controlled by the intramolecular distance between the amino and carboxyl groups in an amino acid structure, although the side-chain group role is minor. The polymerization behavior of Gly reported earlier in the literature is therefore expected to be a typical feature for those of α-amino acids. Equilibrium calculations were conducted to examine effects of dissolved metals as a function of pH on the monomer-polymer equilibria of Gly. Results showed that metals shift the equilibria toward the monomer side, particularly at neutral and alkaline pH. Metals that form weak interaction with Gly (e.g., Mg2+) have no noticeable influence on the polymerization, although strong interaction engenders significant decrease of the equilibrium concentrations of Gly peptides. Considering chemical and structural characteristics of Gly and Gly peptides that control their interactions with metals, it can be expected that similar responses to the addition of metals are applicable in the polymerization of neutral α-amino acids. Neutral and alkaline aqueous environments with dissolved metals having high affinity with neutral α-amino acids (e.g., Cu2+) are therefore not beneficial places for peptide bond formation on the primitive

  20. The pH at the First Equivalence Point in the Titration of a Diprotic Acid

    NASA Astrophysics Data System (ADS)

    Ault, Addison

    2003-12-01

    Some readers will note a similarity between this approach and the one I took in a paper entitled “Do pH in Your Head” (2). In an example in that article the isoelectric pH of glycine (the pH at which the average charge of a glycine molecule is zero), has the value of 6.0, which is exactly half-way between 2.4, the pKa of the carboxyl group of glycine, and 9.6, the pKa of the ammonium group of glycine. This is what one would expect when realizing that a solution of neutral glycine right out of the bottle is equivalent to glycine obtained by titration of the conjugate acid of glycine to the first equivalence point. Those who are interested might want to consider why the isoelectric pH of an “acidic” amino acid, such as alanine, is exactly half-way between the pKa values of the two carboxyl groups, and why the isoelectric pH of a “basic” amino acid such as lysine is exactly half-way between the pKa values of the two ammonium groups.

  1. Polyamine/salt-assembled microspheres coated with hyaluronic acid for targeting and pH sensing.

    PubMed

    Zhang, Pan; Yang, Hui; Wang, Guojun; Tong, Weijun; Gao, Changyou

    2016-06-01

    The poly(allylamine hydrochloride)/trisodium citrate aggregates were fabricated and further covalently crosslinked via the coupling reaction of carboxylic sites on trisodium citrate with the amine groups on polyamine, onto which poly-L-lysine and hyaluronic acid were sequentially assembled, forming stable microspheres. The pH sensitive dye and pH insensitive dye were further labeled to enable the microspheres with pH sensing property. Moreover, these microspheres could be specifically targeted to HeLa tumor cells, since hyaluronic acid can specifically recognize and bind to CD44, a receptor overexpressed on many tumor cells. Quantitative pH measurement by confocal laser scanning microscopy demonstrated that the microspheres were internalized into HeLa cells, and accumulated in acidic compartments. By contrast, only a few microspheres were adhered on the NIH 3T3 cells surface. The microspheres with combined pH sensing property and targeting ability can enhance the insight understanding of the targeted drug vehicles trafficking after cellular internalization. PMID:26954089

  2. Amphipathic β2,2-Amino Acid Derivatives Suppress Infectivity and Disrupt the Intracellular Replication Cycle of Chlamydia pneumoniae

    PubMed Central

    Tiirola, Terttu M.; Strøm, Morten B.; Vuorela, Pia M.

    2016-01-01

    We demonstrate in the current work that small cationic antimicrobial β2,2-amino acid derivatives (Mw < 500 Da) are highly potent against Chlamydia pneumoniae at clinical relevant concentrations (< 5 μM, i.e. < 3.4 μg/mL). C. pneumoniae is an atypical respiratory pathogen associated with frequent treatment failures and persistent infections. This gram-negative bacterium has a biphasic life cycle as infectious elementary bodies and proliferating reticulate bodies, and efficient treatment is challenging because of its long and obligate intracellular replication cycle within specialized inclusion vacuoles. Chlamydicidal effect of the β2,2-amino acid derivatives in infected human epithelial cells was confirmed by transmission electron microscopy. Images of infected host cells treated with our lead derivative A2 revealed affected chlamydial inclusion vacuoles 24 hours post infection. Only remnants of elementary and reticulate bodies were detected at later time points. Neither the EM studies nor resazurin-based cell viability assays showed toxic effects on uninfected host cells or cell organelles after A2 treatment. Besides the effects on early intracellular inclusion vacuoles, the ability of these β2,2-amino acid derivatives to suppress Chlamydia pneumoniae infectivity upon treatment of elementary bodies suggested also a direct interaction with bacterial membranes. Synthetic β2,2-amino acid derivatives that target C. pneumoniae represent promising lead molecules for development of antimicrobial agents against this hard-to-treat intracellular pathogen. PMID:27280777

  3. Influence of sodium chloride, pH, and lactic acid bacteria on anaerobic lactic acid utilization during fermented cucumber spoilage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cucumbers are preserved commercially by natural fermentations in 5% to 8% sodium chloride (NaCl) brines. Occasionally, fermented cucumbers spoil after the primary fermentation is complete. This spoilage has been characterized by decreases in lactic acid and a rise in brine pH caused by microbial ins...

  4. Effects of angiotensin II on intracellular Ca2+ and pH in isolated beating rabbit hearts and myocytes loaded with the indicator indo-1.

    PubMed Central

    Ikenouchi, H; Barry, W H; Bridge, J H; Weinberg, E O; Apstein, C S; Lorell, B H

    1994-01-01

    1. Angiotensin II increases myocardial contractility in several species, including the rabbit and man. However, it is controversial whether the predominant mechanism is an increase in free cytosolic [Ca2+]i or a change in myofilament Ca2+ sensitivity. To address this question, we infused angiotensin II in isolated perfused rabbit hearts loaded with the Ca2+ indicator indo-1 AM and measured changes in beat-to-beat surface transients of the Ca2+i-sensitive 400:500 nm ratio and left ventricular contractility. The effects of angiotensin II were compared with the response to a Ca(2+)-dependent increase in the inotropic state produced by a change in the perfusate [Ca2+] from 0.9 to 3.6 nM. 2. In the isolated beating heart, an increase in perfusate [Ca2+] caused an increase in left ventricular pressure +dP/dt in association with an increase in peak systolic [Ca2+]i. Angiotensin II perfusion caused a similar increase in left ventricular +dP/dt in the absence of any increase in peak systolic [Ca2+]i. 3. To exclude any contribution of non-myocyte sources of Ca(2+)-sensitive fluorescence which may be present in the intact heart, we also compared the effects of angiotensin II and a change in superfusate [Ca2+] in collagenase-dissociated paced adult rabbit ventricular myocytes loaded with indo-1 AM. In the isolated rabbit myocytes a change in perfusate [Ca2+] from 0.9 to 3.6 mM caused an increase in peak systolic cell shortening coincident with an increase in peak systolic [Ca2+]i. In contrast, angiotensin II caused a similar increase in peak systolic cell shortening whereas there was no increase in peak systolic [Ca2+]i. There was also no change in inward Ca2+ current (ICa) in response to angiotensin II. 4. To investigate further the mechanism of the positive inotropic action of angiotensin II, its effects on intracellular pH were studied in isolated rabbit myocytes loaded with the fluorescent H+ probe SNARF 1. These experiments demonstrated that angiotensin II induced a 0.2 pH

  5. Autoinducer-2 detection among commensal oral streptococci is dependent on pH and boric acid.

    PubMed

    Cuadra, Giancarlo A; Frantellizzi, Ashley J; Gaesser, Kimberly M; Tammariello, Steven P; Ahmed, Anika

    2016-07-01

    Autoinducer-2, considered a universal signaling molecule, is produced by many species of bacteria; including oral strains. Structurally, autoinducer-2 can exist bound to boron (borated autoinducer-2). Functionally, autoinducer-2 has been linked to important bacterial processes such as virulence and biofilm formation. In order to test production of autoinducer-2 by a given bacterial strain, a bioassay using marine bioluminescent bacteria Vibrio harveyi as a reporter for autoinducer-2 has been designed. We hypothesize that pH adjustment and addition of boron are required for optimal bioluminescence and accurate autoinducer-2 detection. Using this reporter strain we tested autoinducer-2 activity from two oral commensal species, Streptococcus gordonii DL1 and Streptococcus oralis 34. Spent broth was collected and adjusted to pH 7.5 and supplemented with boric acid prior to measuring autoinducer- 2 activity. Results show that low pH inhibits bioluminescence of the reporter strain, but pH 7.5 allows for bioluminescence induction and proper readings of autoinducer-2 activity. Addition of boric acid also has a positive effect on bioluminescence allowing for a more sensitive detection of autoinducer-2 activity. Our data suggests that although autoinducer-2 is present in spent broth, low pH and/or low levels of boric acid become an obstacle for proper autoinducer-2 detection. For proper autoinducer-2 detection, we propose a protocol using this bioassay to include pH adjustment and boric acid addition to spent broth. Studies on autoinducer-2 activity in several bacteria species represent an important area of study as this universal signaling molecule is involved in critical bacterial phenotypes such as virulence and biofilm formation. PMID:27350615

  6. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization.

    PubMed

    Bates, Ryan C; Fees, Colby P; Holland, William L; Winger, Courtney C; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J

    2014-02-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  7. Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism

    PubMed Central

    Toledo, Daniel A. M.; Roque, Natália R.; Teixeira, Lívia; Milán-Garcés, Erix A.; Carneiro, Alan B.; Almeida, Mariana R.; Andrade, Gustavo F. S.; Martins, Jefferson S.; Pinho, Roberto R.; Freire-de-Lima, Célio G.; Bozza, Patrícia T.; D’Avila, Heloisa

    2016-01-01

    Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas’ disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host. PMID:27490663

  8. Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism.

    PubMed

    Toledo, Daniel A M; Roque, Natália R; Teixeira, Lívia; Milán-Garcés, Erix A; Carneiro, Alan B; Almeida, Mariana R; Andrade, Gustavo F S; Martins, Jefferson S; Pinho, Roberto R; Freire-de-Lima, Célio G; Bozza, Patrícia T; D'Avila, Heloisa; Melo, Rossana C N

    2016-01-01

    Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas' disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host. PMID:27490663

  9. Activation of Src and release of intracellular calcium by phosphatidic acid during Xenopus laevis fertilization

    PubMed Central

    Bates, Ryan C.; Fees, Colby P.; Holland, William L.; Winger, Courtney C.; Batbayar, Khulan; Ancar, Rachel; Bergren, Todd; Petcoff, Douglas; Stith, Bradley J.

    2014-01-01

    We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC- γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca]i). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 minute after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca]i and other fertilization events. As compared to 14 other lipids, PA strongly bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca]i, PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca]i release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca]i release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization. PMID:24269904

  10. Bilayers and wormlike micelles at high pH in fatty acid soap systems.

    PubMed

    Xu, Wenlong; Liu, Huizhong; Song, Aixin; Hao, Jingcheng

    2016-03-01

    Bilayers at high pH in the fatty acid systems of palmitic acid/KOH/H2O, palmitic acid/CsOH/H2O, stearic acid/KOH/H2O and stearic acid/CsOH/H2O can form spontaneously (Xu et al., 2014, 2015). In this work, the bilayers can still be observed at 25°C with an increase of the concentration of fatty acids. We found that wormlike micelles can also be prepared in the fatty acid soap systems at high pH, even though the temperature was increased to be 50°C. The viscoelasticity, apparent viscosity, yield stress of the bilayers were determined by the rheological measurements. Wormlike micelles were identified by cryogenic transmission electron microscopy (cryo-TEM) and emphasized by the rheological characterizations, which are in accordance with the Maxwell fluids with good fit of Cole-Cole plots. The phase transition temperature was determined by differential scanning calorimetry (DSC) and the transition process was recorded. The regulating role of counterions of fatty acids were discussed by (CH3)4N(+), (C2H5)4N(+), (C3H7)4N(+), and (C4H9)4N(+) as comparison, concluding that counterions with appropriate hydrated radius were the vital factor in the formation wormlike micelles. PMID:26688122

  11. Antimicrobial peptides (AMPs) produced by Saccharomyces cerevisiae induce alterations in the intracellular pH, membrane permeability and culturability of Hanseniaspora guilliermondii cells.

    PubMed

    Branco, Patrícia; Viana, Tiago; Albergaria, Helena; Arneborg, Nils

    2015-07-16

    Saccharomyces cerevisiae produces antimicrobial peptides (AMPs) during alcoholic fermentation that are active against several wine-related yeasts (e.g. Hanseniaspora guilliermondii) and bacteria (e.g. Oenococcus oeni). In the present study, the physiological changes induced by those AMPs on sensitive H. guilliermondii cells were evaluated in terms of intracellular pH (pHi), membrane permeability and culturability. Membrane permeability was evaluated by staining cells with propidium iodide (PI), pHi was determined by a fluorescence ratio imaging microscopy (FRIM) technique and culturability by a classical plating method. Results showed that the average pHi of H. guilliermondii cells dropped from 6.5 (healthy cells) to 5.4 (damaged cells) after 20 min of exposure to inhibitory concentrations of AMPs, and after 24 h 77.0% of the cells completely lost their pH gradient (∆pH=pHi-pHext). After 24h of exposure to AMPs, PI-stained (dead) cells increased from 0% to 77.7% and the number of viable cells fell from 1×10(5) to 10 CFU/ml. This means that virtually all cells (99.99%) became unculturable but that a sub-population of 22.3% of the cells remained viable (as determined by PI staining). Besides, pHi results showed that after 24h, 23% of the AMP-treated cells were sub-lethally injured (with 0<∆pH<3). Taken together, these results indicated that this subpopulation was under a viable but non-culturable (VBNC) state, which was further confirmed by recuperation assays. In summary, our study reveals that these AMPs compromise the plasma membrane integrity (and possibly also the vacuole membrane) of H. guilliermondii cells, disturbing the pHi homeostasis and inducing a loss of culturability. PMID:25897995

  12. Recovery of carboxylic acids at pH greater than pK{sub a}

    SciTech Connect

    Tung, L.A.

    1993-08-01

    Economics of producing carboxylic acids by fermentation is often dominated, not by the fermentation cost, but by the cost of recovering and purifying the acids from dilute aqueous solutions. Experiments were performed to measure uptakes of lactic and succinic acids as functions of pH by basic polymeric sorbents; sorbent regeneration was also tested. Performance at pH > pK{sub a} and regenerability depend on sorbent basicity; apparent pK{sub a} and monomer pK{sub a} can be used to predict sorbent performance. Two basic amine extractants, Alamine 336 and Amberlite LA-2, in were also studied; they are able to sustain capacity to higher pH in diluents that stabilize the acid-amine complex through H bonding. Secondary amines perform better than tert-amines in diluents that solvate the additional proton. Competitive sulfate and phosphate, an interference in fermentation, are taken up by sorbents more strongly than by extractants. The third step in the proposed fermentation process, the cracking of the trimethylammonium (TMA) carboxylate, was also examined. Because lactic acid is more soluble and tends to self-esterify, simple thermal cracking does not remove all TMA; a more promising approach is to esterify the TMA lactate by reaction with an alcohol.

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

    SciTech Connect

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

    1986-03-05

    Lizards have been shown to reduce ventilation during CO/sub 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/sub 2/ breathing and made serial measurements of tail (skeletal) muscle pHi, levels of phosphocreatine (PCr), and ATP utilizing high resolution /sup 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<.05) of control. The reduced PCr/ATP ratio does not appear due to decreased O/sub 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.

  14. Overexpression of the cystic fibrosis transmembrane conductance regulator in NIH 3T3 cells lowers membrane potential and intracellular pH and confers a multidrug resistance phenotype.

    PubMed Central

    Wei, L Y; Stutts, M J; Hoffman, M M; Roepe, P D

    1995-01-01

    Because of the similarities between the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance (MDR) proteins, recent observations of decreased plasma membrane electrical potential (delta psi) in cells overexpressing either MDR protein or the CFTR, and the effects of delta psi on passive diffusion of chemotherapeutic drugs, we have analyzed chemotherapeutic drug resistance for NIH 3T3 cells overexpressing different levels of functional CFTR. Three separate clones not previously exposed to chemotherapeutic drugs exhibit resistance to doxorubicin, vincristine, and colchicine that is similar to MDR transfectants not previously exposed to chemotherapeutic drugs. Two other clones expressing lower levels of CFTR are less resistant. As shown previously these clones exhibit decreased plasma membrane delta psi similar to MDR transfectants, but four of five exhibit mildly acidified intracellular pH in contrast to MDR transfectants, which are in general alkaline. Thus the MDR protein and CFTR-mediated MDR phenotypes are distinctly different. Selection of two separate CFTR clones on either doxorubicin or vincristine substantially increases the observed MDR and leads to increased CFTR (but not measurable MDR or MRP) mRNA expression. CFTR overexpressors also exhibit a decreased rate of 3H -vinblastine uptake. These data reveal a new and previously unrecognized consequence of CFTR expression, and are consistent with the hypothesis that membrane depolarization is an important determinant of tumor cell MDR. Images FIGURE 1 FIGURE 3 FIGURE 6 PMID:8519988

  15. The metabolic interaction of cancer cells and fibroblasts - coupling between NAD(P)H and FAD, intracellular pH and hydrogen peroxide.

    PubMed

    Druzhkova, Irina N; Shirmanova, Marina V; Lukina, Maria M; Dudenkova, Varvara V; Mishina, Nataliya M; Zagaynova, Elena V

    2016-05-01

    Alteration in the cellular energy metabolism is a principal feature of tumors. An important role in modifying cancer cell metabolism belongs to the cancer-associated fibroblasts. However, the regulation of their interaction has been poorly studied to date. In this study we monitored the metabolic status of both cell types by using the optical redox ratio and the fluorescence lifetimes of the metabolic co-factors NAD(P)H and FAD, in addition to the intracellular pH and the hydrogen peroxide levels in the cancer cells, using genetically encoded sensors. In the co-culture of human cervical carcinoma cells HeLa and human fibroblasts we observed a metabolic shift from oxidative phosphorylation toward glycolysis in cancer cells, and from glycolysis toward OXPHOS in fibroblasts, starting from Day 2 of co-culturing. The metabolic switch was accompanied by hydrogen peroxide production and slight acidification of the cytosol in the cancer cells in comparison with that of the corresponding monoculture. Therefore, our HeLa-huFb system demonstrated metabolic behavior similar to Warburg type tumors. To our knowledge, this is the first time that these 3 parameters have been investigated together in a model of tumor-stroma co-evolution. We propose that determination of the start-point of the metabolic alterations and understanding of the mechanisms of their realization can open a new ways for cancer treatment. PMID:26986068

  16. Algal and Bacterial Activities in Acidic (pH 3) Strip Mine Lakes

    PubMed Central

    Gyure, Ruth A.; Konopka, Allan; Brooks, Austin; Doemel, William

    1987-01-01

    Reservoir 29 and Lake B are extremely acid lakes (epilimnion pHs of 2.7 and 3.2, respectively), because they receive acidic discharges from coal refuse piles. They differ in that the pH of profundal sediments in Reservoir 29 increased from 2.7 to 3.8 during the period of thermal stratification, whereas permanently anoxic sediments in Lake B had a pH of 6.2. The pH rise in Reservoir 29 sediments was correlated with a temporal increase in H2S concentration in the anaerobic hypolimnion from 0 to >1 mM. The chlorophyll a levels in the epilimnion of Reservoir 29 were low, and the rate of primary production was typical of an oligotrophic system. However, there was a dense 10-cm layer of algal biomass at the bottom of the metalimnion. Production by this layer was low owing to light limitation and possibly H2S toxicity. The specific photosynthetic rates of epilimnetic algae were low, which suggests that nutrient availability is more important than pH in limiting production. The highest photosynthetic rates were obtained in water samples incubated at pH 2.7 to 4. Heterotrophic bacterial activity (measured by [14C]glucose metabolism) was greatest at the sediment/water interface. Bacterial production (assayed by thymidine incorporation) was as high in Reservoir 29 as in a nonacid mesotrophic Indiana lake. PMID:16347430

  17. Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant.

    PubMed

    Hafke, J B; Neff, R; Hütt, M T; Lüttge, U; Thiel, G

    2001-01-01

    In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pHcyt) were monitored in mesophyll protoplasts from the CAM plant Kalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pHcyt indicator. At the beginning of the light phase, pHcyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pHcyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring delta pHcyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H(+)-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pHcyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pHcyt. A diurnal oscillation in pHcyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics. PMID:11732184

  18. Effect of acidic pH on the stability of α-synuclein dimers.

    PubMed

    Lv, Zhengjian; Krasnoslobodtsev, Alexey V; Zhang, Yuliang; Ysselstein, Daniel; Rochet, Jean Christophe; Blanchard, Scott C; Lyubchenko, Yuri L

    2016-10-01

    Environmental factors, such as acidic pH, facilitate the assembly of α-synuclein (α-Syn) in aggregates, but the impact of pH on the very first step of α-Syn aggregation remains elusive. Recently, we developed a single-molecule approach that enabled us to measure directly the stability of α-Syn dimers. Unlabeled α-Syn monomers were immobilized on a substrate, and fluorophore-labeled monomers were added to the solution to allow them to form dimers with immobilized α-Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single-molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of α-Syn dimers. The experiments were performed at pH 5 and 7 for wild-type α-Syn and for two mutants containing familial type mutations E46K and A53T. We demonstrate that a decrease of pH resulted in more than threefold increase in the α-Syn dimers lifetimes with some variability between the α-Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96-140 of α-Syn and this electrostatic effect facilitates the association of the two monomers. Given that dimerization is the first step of α-Syn aggregation, we posit that the electrostatic effect thereby contributes to accelerating α-Syn aggregation at acidic pH. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 715-724, 2016. PMID:27177831

  19. Cellular delivery of quantum dot-bound hybridization probe for detection of intracellular pre-microRNA using chitosan/poly(γ-glutamic acid) complex as a carrier.

    PubMed

    Geng, Yao; Lin, Dajie; Shao, Lijia; Yan, Feng; Ju, Huangxian

    2013-01-01

    A quantum dot (QD)-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP) via Au-S bond and then binding 3'-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics. PMID:23762388

  20. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  1. Anoxic Biodegradation of Isosaccharinic Acids at Alkaline pH by Natural Microbial Communities

    PubMed Central

    Rout, Simon P.; Charles, Christopher J.; Doulgeris, Charalampos; McCarthy, Alan J.; Rooks, Dave J.; Loughnane, J. Paul; Laws, Andrew P.; Humphreys, Paul N.

    2015-01-01

    One design concept for the long-term management of the UK’s intermediate level radioactive wastes (ILW) is disposal to a cementitious geological disposal facility (GDF). Under the alkaline (10.0<pH>13.0) anoxic conditions expected within a GDF, cellulosic wastes will undergo chemical hydrolysis. The resulting cellulose degradation products (CDP) are dominated by α- and β-isosaccharinic acids (ISA), which present an organic carbon source that may enable subsequent microbial colonisation of a GDF. Microcosms established from neutral, near-surface sediments demonstrated complete ISA degradation under methanogenic conditions up to pH 10.0. Degradation decreased as pH increased, with β-ISA fermentation more heavily influenced than α-ISA. This reduction in degradation rate was accompanied by a shift in microbial population away from organisms related to Clostridium sporosphaeroides to a more diverse Clostridial community. The increase in pH to 10.0 saw an increase in detection of Alcaligenes aquatilis and a dominance of hydrogenotrophic methanogens within the Archaeal population. Methane was generated up to pH 10.0 with acetate accumulation at higher pH values reflecting a reduced detection of acetoclastic methanogens. An increase in pH to 11.0 resulted in the accumulation of ISA, the absence of methanogenesis and the loss of biomass from the system. This study is the first to demonstrate methanogenesis from ISA by near surface microbial communities not previously exposed to these compounds up to and including pH 10.0. PMID:26367005

  2. Anoxic Biodegradation of Isosaccharinic Acids at Alkaline pH by Natural Microbial Communities.

    PubMed

    Rout, Simon P; Charles, Christopher J; Doulgeris, Charalampos; McCarthy, Alan J; Rooks, Dave J; Loughnane, J Paul; Laws, Andrew P; Humphreys, Paul N

    2015-01-01

    One design concept for the long-term management of the UK's intermediate level radioactive wastes (ILW) is disposal to a cementitious geological disposal facility (GDF). Under the alkaline (10.0<pH>13.0) anoxic conditions expected within a GDF, cellulosic wastes will undergo chemical hydrolysis. The resulting cellulose degradation products (CDP) are dominated by α- and β-isosaccharinic acids (ISA), which present an organic carbon source that may enable subsequent microbial colonisation of a GDF. Microcosms established from neutral, near-surface sediments demonstrated complete ISA degradation under methanogenic conditions up to pH 10.0. Degradation decreased as pH increased, with β-ISA fermentation more heavily influenced than α-ISA. This reduction in degradation rate was accompanied by a shift in microbial population away from organisms related to Clostridium sporosphaeroides to a more diverse Clostridial community. The increase in pH to 10.0 saw an increase in detection of Alcaligenes aquatilis and a dominance of hydrogenotrophic methanogens within the Archaeal population. Methane was generated up to pH 10.0 with acetate accumulation at higher pH values reflecting a reduced detection of acetoclastic methanogens. An increase in pH to 11.0 resulted in the accumulation of ISA, the absence of methanogenesis and the loss of biomass from the system. This study is the first to demonstrate methanogenesis from ISA by near surface microbial communities not previously exposed to these compounds up to and including pH 10.0. PMID:26367005

  3. Synthesis and characterization of a pH responsive folic acid functionalized polymeric drug delivery system.

    PubMed

    Li, Xia; McTaggart, Matt; Malardier-Jugroot, Cecile

    2016-01-01

    We report the computational analysis, synthesis and characterization of folate functionalized poly(styrene-alt-maleic anhydride), PSMA for drug delivery purpose. The selection of the proper linker between the polymer and the folic acid group was performed before conducting the synthesis using Density Functional Theory (DFT). The computational results showed the bio-degradable linker 2, 4-diaminobutyric acid, DABA as a good candidate allowing flexibility of the folic acid group while maintaining the pH sensitivity of PSMA, used as a trigger for drug release. The synthesis was subsequently carried out in multi-step experimental procedures. The functionalized polymer was characterized using InfraRed spectroscopy, Nuclear Magnetic Resonance and Dynamic Light Scattering confirming both the chemical structure and the pH responsiveness of PSMA-DABA-Folate polymers. This study provides an excellent example of how computational chemistry can be used in selection process for the functional materials and product characterization. The pH sensitive polymers are expected to be used in delivering anti-cancer drugs to solid tumors with overly expressed folic acid receptors. PMID:27183249

  4. Compartmentation of malic acid in mesophyll cells of Kalanchoee daigremontiana: indications of a intracellular cytosolic vesicle transport mechanism

    SciTech Connect

    Balsamo, R.A.; Uribe, E.G.

    1987-04-01

    Leaf tissue was harvested over a 24hr period at one to three hour intervals. The malic acid levels in the tissue were assayed spectrophotometrically and the percent cell volume occupied by cytosolic vesicles in replicate samples was determined. The total volume of the cytosolic vesicles fluctuated throughout the photoperiod concommitantly with malic acid concentrations present in the tissue. An intact leaf tissue section (10.2cm/sup 2/) was radiolabeled with /sup 14/CO/sub 2/ seven hours into the dark period for thirty minutes. Two dimensional thin layer chromatography and electrophoresis of the tissue determined that 96% of the label was incorporated into malic acid. A freeze substitution procedure was initiated followed by microautoradiography (Fisher 1971) which allowed for the tracing of intracellular malic acid migration and compartmentation within the mesophyll cells. The results and interpretation of this experiment will be presented.

  5. Dynamics of fatty acid vesicles in response to pH stimuli.

    PubMed

    Ikari, Keita; Sakuma, Yuka; Jimbo, Takehiro; Kodama, Atsuji; Imai, Masayuki; Monnard, Pierre-Alain; Rasmussen, Steen

    2015-08-21

    We investigate the dynamics of decanoic acid/decanoate (DA) vesicles in response to pH stimuli. Two types of dynamic processes induced by the micro-injection of NaOH solutions are sequentially observed: deformations and topological transitions. In the deformation stage, DA vesicles show a series of shape deformations, i.e., prolate-oblate-stomatocyte-sphere. In the topological transition stage, spherical DA vesicles follow either of the two pathways, pore formation and vesicle fusion. The pH stimuli modify a critical aggregation concentration of DA molecules, which causes the solubilization of DA molecules in the outer leaflet of the vesicle bilayers. This solubilization decreases the outer surface area of the vesicle, thereby increasing surface tension. A kinetic model based on area difference elasticity theory can accurately describe the dynamics of DA vesicles triggered by pH stimuli. PMID:26166464

  6. Acidic pH increases airway surface liquid viscosity in cystic fibrosis.

    PubMed

    Tang, Xiao Xiao; Ostedgaard, Lynda S; Hoegger, Mark J; Moninger, Thomas O; Karp, Philip H; McMenimen, James D; Choudhury, Biswa; Varki, Ajit; Stoltz, David A; Welsh, Michael J

    2016-03-01

    Cystic fibrosis (CF) disrupts respiratory host defenses, allowing bacterial infection, inflammation, and mucus accumulation to progressively destroy the lungs. Our previous studies revealed that mucus with abnormal behavior impaired mucociliary transport in newborn CF piglets prior to the onset of secondary manifestations. To further investigate mucus abnormalities, here we studied airway surface liquid (ASL) collected from newborn piglets and ASL on cultured airway epithelia. Fluorescence recovery after photobleaching revealed that the viscosity of CF ASL was increased relative to that of non-CF ASL. CF ASL had a reduced pH, which was necessary and sufficient for genotype-dependent viscosity differences. The increased viscosity of CF ASL was not explained by pH-independent changes in HCO3- concentration, altered glycosylation, additional pH-induced disulfide bond formation, increased percentage of nonvolatile material, or increased sulfation. Treating acidic ASL with hypertonic saline or heparin largely reversed the increased viscosity, suggesting that acidic pH influences mucin electrostatic interactions. These findings link loss of cystic fibrosis transmembrane conductance regulator-dependent alkalinization to abnormal CF ASL. In addition, we found that increasing Ca2+ concentrations elevated ASL viscosity, in part, independently of pH. The results suggest that increasing pH, reducing Ca2+ concentration, and/or altering electrostatic interactions in ASL might benefit early CF. PMID:26808501

  7. Production of Retrovirus-Based Vectors in Mildly Acidic pH Conditions.

    PubMed

    Holic, Nathalie; Fenard, David

    2016-01-01

    Gene transfer vectors based on retroviridae are increasingly becoming a tool of choice for biomedical research and for the development of biotherapies in rare diseases or cancers. To meet the challenges of preclinical and clinical production, different steps of the production process of self-inactivating γ-retroviral (RVs) and lentiviral vectors (LVs) have been improved (e.g., transfection, media optimization, cell culture conditions). However, the increasing need for mass production of such vectors is still a challenge and could hamper their availability for therapeutic use. Recently, we observed that the use of a neutral pH during vector production is not optimal. The use of mildly acidic pH conditions (pH 6) can increase by two- to threefold the production of RVs and LVs pseudotyped with the vesicular stomatitis virus G (VSV-G) or gibbon ape leukemia virus (GALV) glycoproteins. Here, we describe the production protocol in mildly acidic pH conditions of GALVTR- and VSV-G-pseudotyped LVs using the transient transfection of HEK293T cells and the production protocol of GALV-pseudotyped RVs produced from a murine producer cell line. These protocols should help to achieve higher titers of vectors, thereby facilitating experimental research and therapeutic applications. PMID:27317171

  8. Acidic pH increases airway surface liquid viscosity in cystic fibrosis

    PubMed Central

    Tang, Xiao Xiao; Ostedgaard, Lynda S.; Hoegger, Mark J.; Moninger, Thomas O.; Karp, Philip H.; McMenimen, James D.; Choudhury, Biswa; Varki, Ajit; Stoltz, David A.; Welsh, Michael J.

    2016-01-01

    Cystic fibrosis (CF) disrupts respiratory host defenses, allowing bacterial infection, inflammation, and mucus accumulation to progressively destroy the lungs. Our previous studies revealed that mucus with abnormal behavior impaired mucociliary transport in newborn CF piglets prior to the onset of secondary manifestations. To further investigate mucus abnormalities, here we studied airway surface liquid (ASL) collected from newborn piglets and ASL on cultured airway epithelia. Fluorescence recovery after photobleaching revealed that the viscosity of CF ASL was increased relative to that of non-CF ASL. CF ASL had a reduced pH, which was necessary and sufficient for genotype-dependent viscosity differences. The increased viscosity of CF ASL was not explained by pH-independent changes in HCO3– concentration, altered glycosylation, additional pH-induced disulfide bond formation, increased percentage of nonvolatile material, or increased sulfation. Treating acidic ASL with hypertonic saline or heparin largely reversed the increased viscosity, suggesting that acidic pH influences mucin electrostatic interactions. These findings link loss of cystic fibrosis transmembrane conductance regulator–dependent alkalinization to abnormal CF ASL. In addition, we found that increasing Ca2+ concentrations elevated ASL viscosity, in part, independently of pH. The results suggest that increasing pH, reducing Ca2+ concentration, and/or altering electrostatic interactions in ASL might benefit early CF. PMID:26808501

  9. Effect of pH and organic acids on nitrogen transformations and metal dissolution in soils

    SciTech Connect

    Fu, Minhong.

    1989-01-01

    The effect of pH (4, 6, and 8) on nitrogen mineralization was evaluated in three Iowa surface soils treated with crop residues (corn (Zea mays L.), soybean (Glycine max (L.) Merr.), and sorghum (Sorghum vulgare Pers.), or alfalfa (Medicago sativa L.)) and incubated in leaching columns under aerobic conditions at 30C for 20 weeks. In general, N mineralization was significantly depressed at soil pH 4, compared with pH 6 or 8. The types of crop residues added influenced the pattern and amount of N mineralization. A study on the effect of 19 trace elements on the nitrate red activity of four Iowa surface soils showed that most trace elements inhibited this enzyme in acid and neutral soils. The trace elements Ag(I), Cd(II), Se(IV), As(V), and W(VI) were the most effective inhibitors, with >75% inhibition. Mn(II) was the least effective inhibitor, with <10% inhibition. Other trace elements included Cu(I), Co(II), Cu(II), Fe(II), Ni(II), Pb(II), Zn(II), Al(III), As(III), Cr(III), Fe(III), V(IV), Mo(VI), and Se(VI). The application of high-performance liquid chromatography (HPLC) showed that, when coupled to a refractive index detector, it is a rapid, sensitive, and accurate method for determining organic acids in soils. Three organic acids, acetic (2-20 mM), propionic (0-3 mM), and n-butyric (0-1.4 mM), were identified with HPLC and confirmed by gas chromatography in crop-residue-treated soils incubated under waterlogged conditions at 25C for 72 h. No organic acids were detected under aerobic conditions. Four mineral acids and 29 organic acids were studied for their effect on N mineralization and metal dissolution in soils incubated under waterlogged conditions at 30C for 10 days.

  10. Chlorogenic acid increased 5-hydroxymethylfurfural formation when heating fructose alone or with aspartic acid at two pH levels.

    PubMed

    Zhang, Zhenhua; Zou, Yueyu; Wu, Taigang; Huang, Caihuan; Pei, Kehan; Zhang, Guangwen; Lin, Xiaohua; Bai, Weibin; Ou, Shiyi

    2016-01-01

    Chlorogenic acid (CGA) is a phenolic acid that ubiquitously exists in fruits. This work aims to investigate whether and how CGA influences HMF formation during heating fructose alone, or with an amino acid. The results showed that that CGA increased 5-hydroxymethylfurfural (HMF) formation. At pH 5.5 and 7.0, the addition of 5.0 μmol/ml CGA increased HMF formation by 49.4% and 25.2%, respectively when heating fructose alone, and by 9.0% and 16.7%, respectively when heating fructose with aspartic acid. CGA significantly increased HMF formation by promoting 3-deoxosone formation, and its conversion to HMF by inhibiting HMF elimination, especially in the Maillard reaction system. A comparison of the catalytic capacity of CGA with its six analogous compounds showed that both its di-hydroxyphenyl and carboxyl groups function in increasing HMF formation. PMID:26213045

  11. GABAAergic stimulation modulates intracellular protein arginine methylation.

    PubMed

    Denman, Robert B; Xie, Wen; Merz, George; Sung, Ying-Ju

    2014-06-20

    Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na(+)/H(+) antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2β2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome. PMID:24793772

  12. Self-assembly of humic acid: influence of pH and chemical composition

    NASA Astrophysics Data System (ADS)

    Chilom, G.; Nagy, Z.; Delp, S.; Huff, G.; Rice, J. A.

    2010-12-01

    Interest in enhancing the residence time of soil organic matter (SOM) through natural or engineered mechanisms as a possible means of sequestering organic carbon to mitigate the impacts of carbon-dioxide induced global warming effects has steadily increased over the last decade. Humic substances are major organic constituents of SOM and were recently shown that can self-organize or self-assemble into a composite material with different characteristics than those of the starting materials, and the organized state controls its mineralization by microorganisms. This study examines the role of pH and the relative concentration of humic-like amphiphilic (HA2) and lipid-like (L1) components in the self-assembly of the lipid-humic composite (L0). The L0, L1 and HA2 fractions were isolated using a combination of organic solvent and aqueous alkaline extractions from two humic acid samples at various pH values. HA2 and L1 isolated at low pH were mixed in various mass ratios in organic solvent in order to “reassemble” L0. The data show that the amount of L0 decreased with increasing the pH and reached a constant value from pH 6 to pH 11, and the proportion of L1 increased with the pH. Comparative measurements of the specific heat capacity as a function of temperature of the recombined L0 reveal differences when compared to the physical mixture of the HA2 and L1 depending on the ratio of the components. These differences are an indication that the recombined L0’s solid-state structure is more than just a mixture of components and is determined by specific interactions between its components.

  13. Indomethacin inhibits tetrodotoxin-resistant Na(+) channels at acidic pH in rat nociceptive neurons.

    PubMed

    Nakamura, Michiko; Jang, Il-Sung

    2016-06-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are well-known inhibitors of cyclooxygenases (COXs) and are widely used for the treatment of inflammatory pain; however several NSAIDs display COX-independent analgesic action including the inhibition of voltage-gated Na(+) channels expressed in primary afferent neurons. In the present study, we examined whether NSAIDs modulate tetrodotoxin-resistant (TTX-R) Na(+) channels and if this modulation depends on the extracellular pH. The TTX-R Na(+) currents were recorded from small-sized trigeminal ganglion neurons by using a whole-cell patch clamp technique. Among eight NSAIDs tested in this study, several drugs, including aspirin and ibuprofen, did not affect TTX-R Na(+) channels either at pH 7.4 or at pH 6.0. However, we found that indomethacin, and, to a lesser extent, ibuprofen and naproxen potently inhibited the peak amplitude of TTX-R Na(+) currents at pH 6.0. The indomethacin-induced inhibition of TTX-R Na(+) channels was more potent at depolarized membrane potentials. Indomethacin significantly shifted both the voltage-activation and voltage-inactivation relationships to depolarizing potentials at pH 6.0. Indomethacin accelerated the development of inactivation and retarded the recovery from inactivation of TTX-R Na(+) channels at pH 6.0. Given that indomethacin and several other NSAIDs could further suppress local nociceptive signals by inhibiting TTX-R Na(+) channels at an acidic pH in addition to the classical COX inhibition, these drugs could be particularly useful for the treatment of inflammatory pain. PMID:26898291

  14. Proton-dependent zinc release from intracellular ligands

    PubMed Central

    Kiedrowski, Lech

    2014-01-01

    In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn2+ into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn2+. Although exposure to the protonophore FCCP precludes re-loading of the mitochondria and acidic organelles with Zn2+, FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn2+. Therefore, Zn2+-releasing stores were not mitochondria or acidic organelles but rather intracellular Zn2+ ligands. To test which ligands might be involved, the rate of acid-induced Zn2+ release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn2+] was monitored in vitro using the ratiometric Zn2+-sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn2+ but did chelate Cu2+; the remaining ligands chelated Zn2+ and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn2+ release. The zinc-cysteine complexes also released Zn2+ when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn2+, these complexes may represent the stores responsible for an acid-induced intracellular Zn2+ release. PMID:24606401

  15. The Cytosolic pH of Individual Saccharomyces cerevisiae Cells Is a Key Factor in Acetic Acid Tolerance

    PubMed Central

    Fernández-Niño, Miguel; Marquina, Maribel; Swinnen, Steve; Rodríguez-Porrata, Boris

    2015-01-01

    It was shown recently that individual cells of an isogenic Saccharomyces cerevisiae population show variability in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the population level. In the current study, we investigated whether cell-to-cell variability in acetic acid tolerance could be explained by the observed differences in the cytosolic pHs of individual cells immediately before exposure to the acid. Results obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic pH drop after exposure to the acid. Moreover, only cells with a low initial cytosolic pH, which experienced a less severe drop in cytosolic pH, were able to proliferate. A similar correlation between initial cytosolic pH and cytosolic pH drop was also observed in the more acid-tolerant strain MUCL 11987-9. Interestingly, a fraction of cells in the MUCL 11987-9 population showed initial cytosolic pH values below the minimal cytosolic pH detected in cells of the strain CEN.PK113-7D; consequently, these cells experienced less severe drops in cytosolic pH. Although this might explain in part the difference between the two strains with regard to the number of cells that resumed proliferation, it was observed that all cells from strain MUCL 11987-9 were able to proliferate, independently of their initial cytosolic pH. Therefore, other factors must also be involved in the greater ability of MUCL 11987-9 cells to endure strong drops in cytosolic pH. PMID:26341199

  16. Sensitivity of greenback cutthroat trout to acidic pH and elevated aluminum

    SciTech Connect

    Woodward, D.F. ); Farag, A.M. ); Little E.E.; Steadman, B. ); Yancik, R. )

    1991-01-01

    The greenback cutthroat trout Oncorhynchus clarki stomias is a threatened subspecies native to the upper South Platte and Arkansas rivers between Denver and Fort Collins, Colorado, an area also susceptible to acid deposition. In laboratory studies, the authors exposed this subspecies to nominal pHs of 4.5-6.5 and to nominal aluminum concentrations of 0, 50, 100, and 300 {mu}g/L; the control was pH 6.5 treatment without Al. The authors used soft water that contained 1.3 mg Ca/L. Exposures of 7 days each were made for four early life stages: fertilized egg, eyed embryo, alevin, and swim-up larva. Effects were measured at the end of exposure and again after a recovery period lasting until 40 days posthatch. The alevin stage was the most sensitive: at pH 5.0 with no Al, survival was reduced by 68% and swimming duration by 76%, at pH 6.0 and 50 {mu}g Al/L, swimming duration was reduced by 62%, but survival was not affected. Reductions in whole-body concentrations of Na, K, and Ca indicated organism stress. Sodium was reduced most-about 50% in alevins exposed to pH 5.0 without Al and to pH 6.0 with 50 {mu}g Al/L. Growth and the ratio of RNA to DNA were not affected by any exposure. All responses that were affected during exposure returned to normal by 40 days posthatch. Overall, it appeared that pH 6.0 and 50 {mu}g Al/L might be detrimental to greenback cutthroat trout populations.

  17. Improved volatile fatty acids anaerobic production from waste activated sludge by pH regulation: Alkaline or neutral pH?

    PubMed

    Ma, Huijun; Chen, Xingchun; Liu, He; Liu, Hongbo; Fu, Bo

    2016-02-01

    In this study, the anaerobic fermentation was carried out for volatile fatty acids (VFAs) production at different pH (between 7.0 and 10.0) conditions with untreated sludge and heat-alkaline pretreated waste activated sludge. In the fermentation with untreated sludge, the extent of hydrolysis of organic matters and extent of acidification at alkaline pH are 54.37% and 30.37%, respectively, resulting in the highest VFAs yield at 235.46mg COD/gVS of three pH conditions. In the fermentation with heat-alkaline pretreated sludge, the acidification rate and VFAs yield at neutral pH are 30.98% and 240.14mg COD/gVS, respectively, which are higher than that at other pH conditions. With the glucose or bovine serum albumin as substrate for VFAs production, the neutral pH showed a higher VFAs concentration than the alkaline pH condition. The results of terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that the alkaline pH caused low microbial richness. Based on the results in this study, we demonstrated that the alkaline pH is favor of hydrolysis of organic matter in sludge while neutral pH improved the acidogenesis for the VFAs production from sludge. Our finding is obvious different to the previous research and helpful for the understanding of how heat-alkaline pretreatment and alkaline fermentation influence the VFAs production, and beneficial to the development of VFAs production process. PMID:26652215

  18. Isoelectric focusing of dansylated amino acids in immobilized pH gradients

    NASA Technical Reports Server (NTRS)

    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.

  19. Spontaneous aggregation of humic acid observed with AFM at different pH.

    PubMed

    Colombo, Claudio; Palumbo, Giuseppe; Angelico, Ruggero; Cho, Hyen Goo; Francioso, Ornella; Ertani, Andrea; Nardi, Serenella

    2015-11-01

    Atomic force microscopy in contact (AFM-C) mode was used to investigate the molecular dynamics of leonardite humic acid (HA) aggregate formed at different pH values. HA nanoparticles dispersed at pH values ranging from 2 to 12 were observed on a mica surface under dry conditions. The most clearly resolved and well-resulted AFM images of single particle were obtained at pH 5, where HA appeared as supramolecular particles with a conic shape and a hole in the centre. Those observations suggested that HA formed under these conditions exhibited a pseudo-amphiphilic nature, with secluded hydrophobic domains and polar subunits in direct contact with hydrophilic mica surface. Based on molecular simulation methods, a lignin-carbohydrate complex (LCC) model was proposed to explain the HA ring-like morphology. The LCC model optimized the parameters of β-O-4 linkages between 14 units of 1-4 phenyl propanoid, and resulted in an optimized structure comprising 45-50 linear helical molecules looped spirally around a central cavity. Those results added new insights on the adsorption mechanism of HA on polar surfaces as a function of pH, which was relevant from the point of view of natural aggregation in soil environment. PMID:26295541

  20. Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

    PubMed

    Meza, Beatriz; de-Bashan, Luz E; Bashan, Yoav

    2015-01-01

    Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris. PMID:25554489

  1. Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH.

    PubMed

    Broadbent, J R; Oberg, T S; Hughes, J E; Ward, R E; Brighton, C; Welker, D L; Steele, J L

    2014-03-01

    Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid's pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8. PMID:24370881

  2. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted. PMID:19836400

  3. Catalysis of Glyceraldehyde Synthesis by Primary or Secondary Amino Acids Under Prebiotic Conditions as a Function of pH

    NASA Astrophysics Data System (ADS)

    Breslow, Ronald; Ramalingam, Vijayakumar; Appayee, Chandrakumar

    2013-10-01

    The synthesis of an excess of D-glyceraldehyde by coupling glycolaldehyde with formaldehyde under prebiotic conditions is catalyzed by L amino acids having primary amino groups at acidic pH's, but at neutral or higher pH's they preferentially form L-glyceraldehyde. L Amino acids having secondary amino groups, such as proline, have the reverse preferences, affording excess L-glyceraldehyde at low pH but excess D-glyceraldehyde at higher pHs. Detailed mechanistic proposals make these preferences understandable. The relevance of these findings to the origin of D sugars on prebiotic Earth is described.

  4. pH-Sensitive Polymeric Micelle-based pH Probe for Detecting and Imaging Acidic Biological Environments

    PubMed Central

    Lee, Young Ju; Kang, Han Chang; Hu, Jun; Nichols, Joseph W.; Jeon, Yong Sun; Bae, You Han

    2012-01-01

    To overcome the limitations of monomeric pH probes for acidic tumor environments, this study designed a mixed micelle pH probe composed of polyethylene glycol (PEG)-b- poly(L-histidine) (PHis) and PEG-b-poly(L-lactic acid) (PLLA), which is well-known as an effective antitumor drug carrier. Unlike monomeric histidine and PHis derivatives, the mixed micelles can be structurally destabilized by changes in pH, leading to a better pH sensing system in nuclear magnetic resonance (NMR) techniques. The acidic pH-induced transformation of the mixed micelles allowed pH detection and pH mapping of 0.2–0.3 pH unit differences by pH-induced “on/off”-like sensing of NMR and magnetic resonance spectroscopy (MRS). The micellar pH probes sensed pH differences in non-biological phosphate buffer and biological buffers such as cell culture medium and rat whole blood. In addition, the pH-sensing ability of the mixed micelles was not compromised by loaded doxorubicin. In conclusion, PHis-based micelles could have potential as a tool to simultaneously treat and map the pH of solid tumors in vivo. PMID:22861824

  5. Biogenic precipitation of manganese oxides and enrichment of heavy metals at acidic soil pH

    NASA Astrophysics Data System (ADS)

    Mayanna, Sathish; Peacock, Caroline L.; Schäffner, Franziska; Grawunder, Anja; Merten, Dirk; Kothe, Erika; Büchel, Georg

    2014-05-01

    The precipitation of biogenic Mn oxides at acidic pH is rarely reported and poorly understood, compared to biogenic Mn oxide precipitation at near neutral conditions. Here we identified and investigated the precipitation of biogenic Mn oxides in acidic soil, and studied their role in the retention of heavy metals, at the former uranium mining site of Ronneburg, Germany. The site is characterized by acidic pH, low carbon content and high heavy metal loads including rare earth elements. Specifically, the Mn oxides were present in layers identified by detailed soil profiling and within these layers pH varied from 4.7 to 5.1, Eh varied from 640 to 660 mV and there were enriched total metal contents for Ba, Ni, Co, Cd and Zn in addition to high Mn levels. Using electron microprobe analysis, synchrotron X-ray diffraction and X-ray absorption spectroscopy, we identified poorly crystalline birnessite (δ-MnO2) as the dominant Mn oxide in the Mn layers, present as coatings covering and cementing quartz grains. With geochemical modelling we found that the environmental conditions at the site were not favourable for chemical oxidation of Mn(II), and thus we performed 16S rDNA sequencing to isolate the bacterial strains present in the Mn layers. Bacterial phyla present in the Mn layers belonged to Firmicutes, Actinobacteria and Proteobacteria, and from these phyla we isolated six strains of Mn(II) oxidizing bacteria and confirmed their ability to oxidise Mn(II) in the laboratory. The biogenic Mn oxide layers act as a sink for metals and the bioavailability of these metals was much lower in the Mn layers than in adjacent layers, reflecting their preferential sorption to the biogenic Mn oxide. In this presentation we will report our findings, concluding that the formation of natural biogenic poorly crystalline birnessite can occur at acidic pH, resulting in the formation of a biogeochemical barrier which, in turn, can control the mobility and bioavailability of heavy metals in

  6. Sulfate reduction at low pH to remediate acid mine drainage.

    PubMed

    Sánchez-Andrea, Irene; Sanz, Jose Luis; Bijmans, Martijn F M; Stams, Alfons J M

    2014-03-30

    Industrial activities and the natural oxidation of metallic sulfide-ores produce sulfate-rich waters with low pH and high heavy metals content, generally termed acid mine drainage (AMD). This is of great environmental concern as some heavy metals are highly toxic. Within a number of possibilities, biological treatment applying sulfate-reducing bacteria (SRB) is an attractive option to treat AMD and to recover metals. The process produces alkalinity, neutralizing the AMD simultaneously. The sulfide that is produced reacts with the metal in solution and precipitates them as metal sulfides. Here, important factors for biotechnological application of SRB such as the inocula, the pH of the process, the substrates and the reactor design are discussed. Microbial communities of sulfidogenic reactors treating AMD which comprise fermentative-, acetogenic- and SRB as well as methanogenic archaea are reviewed. PMID:24444599

  7. Influence of pH, bleaching agents, and acid etching on surface wear of bovine enamel

    PubMed Central

    Soares, Ana Flávia; Bombonatti, Juliana Fraga Soares; Alencar, Marina Studart; Consolmagno, Elaine Cristina; Honório, Heitor Marques; Mondelli, Rafael Francisco Lia

    2016-01-01

    ABSTRACT Development of new materials for tooth bleaching justifies the need for studies to evaluate the changes in the enamel surface caused by different bleaching protocols. Objective The aim of this study was to evaluate the bovine dental enamel wear in function of different bleaching gel protocols, acid etching and pH variation. Material and Methods Sixty fragments of bovine teeth were cut, obtaining a control and test areas. In the test area, one half received etching followed by a bleaching gel application, and the other half, only the bleaching gel. The fragments were randomly divided into six groups (n=10), each one received one bleaching session with five hydrogen peroxide gel applications of 8 min, activated with hybrid light, diode laser/blue LED (HL) or diode laser/violet LED (VHL) (experimental): Control (C); 35% Total Blanc Office (TBO35HL); 35% Lase Peroxide Sensy (LPS35HL); 25% Lase Peroxide Sensy II (LPS25HL); 15% Lase Peroxide Lite (LPL15HL); and 10% hydrogen peroxide (experimental) (EXP10VHL). pH values were determined by a pHmeter at the initial and final time periods. Specimens were stored, subjected to simulated brushing cycles, and the superficial wear was determined (μm). ANOVA and Tukey´s tests were applied (α=0.05). Results The pH showed a slight decrease, except for Group LPL15HL. Group LPS25HL showed the highest degree of wear, with and without etching. Conclusion There was a decrease from the initial to the final pH. Different bleaching gels were able to increase the surface wear values after simulated brushing. Acid etching before bleaching increased surface wear values in all groups. PMID:27008254

  8. Mycorrhizal Response to Experimental pH and P Manipulation in Acidic Hardwood Forests

    PubMed Central

    Kluber, Laurel A.; Carrino-Kyker, Sarah R.; Coyle, Kaitlin P.; DeForest, Jared L.; Hewins, Charlotte R.; Shaw, Alanna N.; Smemo, Kurt A.; Burke, David J.

    2012-01-01

    Many temperate forests of the Northeastern United States and Europe have received significant anthropogenic acid and nitrogen (N) deposition over the last century. Although temperate hardwood forests are generally thought to be N-limited, anthropogenic deposition increases the possibility of phosphorus (P) limiting productivity in these forest ecosystems. Moreover, inorganic P availability is largely controlled by soil pH and biogeochemical theory suggests that forests with acidic soils (i.e., <pH 5) are particularly vulnerable to P limitation. Results from previous studies in these systems are mixed with evidence both for and against P limitation. We hypothesized that shifts in mycorrhizal colonization and community structure help temperate forest ecosystems overcome an underlying P limitation by accessing mineral and organic P sources that are otherwise unavailable for direct plant uptake. We examined arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) communities and soil microbial activity in an ecosystem-level experiment where soil pH and P availability were manipulated in mixed deciduous forests across eastern Ohio, USA. One year after treatment initiation, AM root biomass was positively correlated with the most available P pool, resin P, while AM colonization was negatively correlated. In total, 15,876 EcM root tips were identified and assigned to 26 genera and 219 operational taxonomic units (97% similarity). Ectomycorrhizal richness and root tip abundance were negatively correlated with the moderately available P pools, while the relative percent of tips colonized by Ascomycetes was positively correlated with soil pH. Canonical correspondence analysis revealed regional, but not treatment, differences in AM communities, while EcM communities had both treatment and regional differences. Our findings highlight the complex interactions between mycorrhizae and the soil environment and further underscore the fact that mycorrhizal communities do not merely

  9. Influence of pH, bleaching agents, and acid etching on surface wear of bovine enamel.

    PubMed

    Soares, Ana Flávia; Bombonatti, Juliana Fraga Soares; Alencar, Marina Studart; Consolmagno, Elaine Cristina; Honório, Heitor Marques; Mondelli, Rafael Francisco Lia

    2016-02-01

    Development of new materials for tooth bleaching justifies the need for studies to evaluate the changes in the enamel surface caused by different bleaching protocols. Objective The aim of this study was to evaluate the bovine dental enamel wear in function of different bleaching gel protocols, acid etching and pH variation. Material and Methods Sixty fragments of bovine teeth were cut, obtaining a control and test areas. In the test area, one half received etching followed by a bleaching gel application, and the other half, only the bleaching gel. The fragments were randomly divided into six groups (n=10), each one received one bleaching session with five hydrogen peroxide gel applications of 8 min, activated with hybrid light, diode laser/blue LED (HL) or diode laser/violet LED (VHL) (experimental): Control (C); 35% Total Blanc Office (TBO35HL); 35% Lase Peroxide Sensy (LPS35HL); 25% Lase Peroxide Sensy II (LPS25HL); 15% Lase Peroxide Lite (LPL15HL); and 10% hydrogen peroxide (experimental) (EXP10VHL). pH values were determined by a pHmeter at the initial and final time periods. Specimens were stored, subjected to simulated brushing cycles, and the superficial wear was determined (μm). ANOVA and Tukey´s tests were applied (α=0.05). Results The pH showed a slight decrease, except for Group LPL15HL. Group LPS25HL showed the highest degree of wear, with and without etching. Conclusion There was a decrease from the initial to the final pH. Different bleaching gels were able to increase the surface wear values after simulated brushing. Acid etching before bleaching increased surface wear values in all groups. PMID:27008254

  10. Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level

    PubMed Central

    Duan, Xiangke; Li, Yunsong; Du, Qinglin; Huang, Qinqin; Guo, Siyao; Xu, Mengmeng; Lin, Yanping; Liu, Zhidong; Xie, Jianping

    2016-01-01

    Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance. PMID:26806099

  11. Mycobacterium Lysine ε-aminotransferase is a novel alarmone metabolism related persister gene via dysregulating the intracellular amino acid level.

    PubMed

    Duan, Xiangke; Li, Yunsong; Du, Qinglin; Huang, Qinqin; Guo, Siyao; Xu, Mengmeng; Lin, Yanping; Liu, Zhidong; Xie, Jianping

    2016-01-01

    Bacterial persisters, usually slow-growing, non-replicating cells highly tolerant to antibiotics, play a crucial role contributing to the recalcitrance of chronic infections and treatment failure. Understanding the molecular mechanism of persister cells formation and maintenance would obviously inspire the discovery of new antibiotics. The significant upregulation of Mycobacterium tuberculosis Rv3290c, a highly conserved mycobacterial lysine ε-aminotransferase (LAT) during hypoxia persistent model, suggested a role of LAT in persistence. To test this, a lat deleted Mycobacterium smegmatis was constructed. The expression of transcriptional regulator leucine-responsive regulatory protein (LrpA) and the amino acids abundance in M. smegmatis lat deletion mutants were lowered. Thus, the persistence capacity of the deletion mutant was impaired upon norfloxacin exposure under nutrient starvation. In summary, our study firstly reported the involvement of mycobacterium LAT in persister formation, and possibly through altering the intracellular amino acid metabolism balance. PMID:26806099

  12. Life at high salt concentrations, intracellular KCl concentrations, and acidic proteomes

    PubMed Central

    Oren, Aharon

    2013-01-01

    Extremely halophilic microorganisms that accumulate KCl for osmotic balance (the Halobacteriaceae, Salinibacter) have a large excess of acidic amino acids in their proteins. This minireview explores the occurrence of acidic proteomes in halophiles of different physiology and phylogenetic affiliation. For fermentative bacteria of the order Halanaerobiales, known to accumulate KCl, an acidic proteome was predicted. However, this is not confirmed by genome analysis. The reported excess of acidic amino acids is due to a high content of Gln and Asn, which yield Glu and Asp upon acid hydrolysis. The closely related Halorhodospira halophila and Halorhodospira halochloris use different strategies to cope with high salt. The first has an acidic proteome and accumulates high KCl concentrations at high salt concentrations; the second does not accumulate KCl and lacks an acidic proteome. Acidic proteomes can be predicted from the genomes of some moderately halophilic aerobes that accumulate organic osmotic solutes (Halomonas elongata, Chromohalobacter salexigens) and some marine bacteria. Based on the information on cultured species it is possible to understand the pI profiles predicted from metagenomic data from hypersaline environments. PMID:24204364

  13. Cloning and Characterization of an Intracellular Esterase from the Wine-Associated Lactic Acid Bacterium Oenococcus oeni▿ †

    PubMed Central

    Sumby, Krista M.; Matthews, Angela H.; Grbin, Paul R.; Jiranek, Vladimir

    2009-01-01

    We report the cloning and characterization of EstB28, the first esterase to be so characterized from the wine-associated lactic acid bacterium, Oenococcus oeni. The published sequence for O. oeni strain PSU-1 was used to identify putative esterase genes and design PCR primers in order to amplify the corresponding region from strain Ooeni28, an isolate intended for inoculation of wines. In this way a 912-bp open reading frame (ORF) encoding a putative esterase of 34.5 kDa was obtained. The amino acid sequence indicated that EstB28 is a member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid bacteria. This ORF was cloned into Escherichia coli using an appropriate expression system, and the recombinant esterase was purified. Characterization of EstB28 revealed that the optimum temperature, pH, and ethanol concentration were 40°C, pH 5.0, and 28% (vol/vol), respectively. EstB28 also retained marked activity under conditions relevant to winemaking (10 to 20°C, pH 3.5, 14% [vol/vol] ethanol). Kinetic constants were determined for EstB28 with p-nitrophenyl (pNP)-linked substrates ranging in chain length from C2 to C18. EstB28 exhibited greatest specificity for C2 to C4 pNP-linked substrates. PMID:19734337

  14. [Intracellular and extracellular functions of phosphorus compound in the body].

    PubMed

    Segawa, Hiroko; Hanazaki, Ai; Miyamoto, Ken-ichi

    2016-02-01

    Phosphorus, as a phosphate is a component of bone, cellular membrane, and also high-energy phosphate compounds, and nucleic acids. Also phosphate acts as a buffer to maintain the pH and is concerned with functional regulation of several proteins and intracellular signaling through the phosphorylation/dephosphorylation. Thus phosphorus plays a variety of important roles intracellular and extracellular component. A disorder of phosphate homeostasis results bone disorder and general metabolic dysfunction of all body tissues and organs. PMID:26813497

  15. EFFECTS OF PH, SOLID/SOLUTION RATIO, IONIC STRENGTH, AND ORGANIC ACIDS ON PB AND CD ON KAOLINITE

    EPA Science Inventory

    Potentiometric and ion-selective electrode titrations together with batch sorption/desorption experiments, were performed to explain the aqueous and surface complexation reactions between kaolinite, Pb, Cd and organic acids. Variables included pH, ionic strength, metal concentrat...

  16. The capacity of biochar made from common reeds to neutralise pH and remove dissolved metals in acid drainage.

    PubMed

    Mosley, Luke M; Willson, Philip; Hamilton, Benjamin; Butler, Greg; Seaman, Russell

    2015-10-01

    We tested the capacity of biochar (made at 450 °C from a common reed species) to neutralise pH and remove metals in two acid drainage waters (pH 2.6 and 4.6) using column leaching and batch mixing experiments. In the column experiments, the acid drainage water was neutralised upon passage through the biochar with substantial increases (4-5 pH units) in the leachate pH. In the batch experiments, the leachate pH remained above 6.5 when the drainage:biochar ratio was less than approximately 700:1 (L acid drainage:kg biochar) and 20:1 for the pH 4.6 and pH 2.6 drainage waters, respectively. Dissolved metal concentrations were reduced by 89-98 % (Fe ≈ Al > Ni ≈ Zn > Mn) in the leachate from the biochar. A key mechanism of pH neutralisation appears to be solid carbonate dissolution as calcite (CaCO3) was identified (via X-ray diffraction) in the biochar prior to contact with acid drainage, and dissolved alkalinity and Ca was observed in the leachate. Proton and metal removal by cation exchange, direct binding to oxygen-containing functional groups, and metal oxide precipitation also appears important. Further evaluation of the treatment capacity of other biochars and field trials are warranted. PMID:26004563

  17. Influence of five neutralizing products on intra-oral pH after rinsing with simulated gastric acid.

    PubMed

    Lindquist, Birgitta; Lingström, Peter; Fändriks, Lars; Birkhed, Dowen

    2011-08-01

    The aetiology of dental erosion may be of both extrinsic and intrinsic origin. The aim of the present study was to test the ability of various neutralizing products to raise the low intra-oral pH after an erosive exposure, in this case to gastric acid, which was simulated using hydrochloric acid (HCl). Eleven adults participated. They rinsed with 10 ml of 10 mM HCl (pH 2) or 10 ml of 100 mM HCl (pH 1) for 1 min, after which the pH was measured intra-orally for up to 30 min at four sites (two approximal, one buccal, and the dorsum of the tongue). After rinsing with the two acid solutions (pH 1 and pH 2), the following products were used: (i) antacid tablet; (ii) gum arabic lozenge; (iii) mineral water; (iv) milk; and (v) tap water (positive control). The negative control was no product use. The five test products were used for 2 min after the erosive challenge. All the products produced an initially higher pH compared with the negative control. The antacid tablet resulted in the greatest and most rapid increase in pH, followed by the lozenge. In dental practice, the use of any of the neutralizing products tested, especially the antacid tablet, could be recommended in order to increase the intra-oral pH after an erosive challenge. PMID:21726291

  18. pH dependence of methyl phosphonic acid, dipicolinic acid, and cyanide by surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Farquharson, Stuart; Gift, Alan; Maksymiuk, Paul; Inscore, Frank E.; Smith, Wayne W.

    2004-03-01

    U.S. and Coalition forces fighting terrorism in Afghanistan and Iraq must consider a wide range of attack scenarios in addition to car bombings. Among these is the intentional poisoning of water supplies to obstruct military operations. To counter such attacks, the military is developing portable analyzers that can identify and quantify potential chemical agents in water supplies at microgram per liter concentrations within 10 minutes. To aid this effort we have been investigating the value of a surface-enhanced Raman spectroscopy based portable analyzer. In particular we have been developing silver-doped sol-gels to generate SER spectra of chemical agents and their hydrolysis products. Here we present SER spectra of methyl phosphonic acid and cyanide as a function of pH, an important factor affecting quantitation measurements, which to our knowledge has not been examined. In addition, dipicolinic acid, a chemical signature associated with anthrax-causing spores, is also presented.

  19. The role of low molecular weight organic acids on controlling pH in coastal sea water

    NASA Astrophysics Data System (ADS)

    Ding, H.

    2015-12-01

    Series investigation of the Jiaozhou Bay, China, observed existences of three low molecular weight organic acids (LMWOAs), including lactic acid, acetic acid and formic acid, with high concentration in the sea water. Generally, their amount accounted for about 20% of DOC in the sea water of the bay. Human activities around the bay were considered as the major source of the LMWOAs. Also, long term detection showed that the pH value in the Jiaozhou Bay was lower than that in the adjacent Yellow Sea. On average, the difference of pH values between the bay and the Yellow was about 0.2. Due to higher concentrations of the LMWOAs, their contribution to lower pH value of the bay should not be ignored. To validate the effect of LMWOAs on the pH value of the bay, a new software was developed to calculate the pH value in the sea water samples based on alkalinity by adding three items of the three organic acids in the expression. Compared to the traditional pH calculating software, the new software could improve the calculating results significantly. Our results confirmed that LMWOAs was an important control factor to adjust pH values in coastal area.

  20. The effect of pH on the toxicity of fatty acids and fatty acid amides to rainbow trout gill cells.

    PubMed

    Bertin, Matthew J; Voronca, Delia C; Chapman, Robert W; Moeller, Peter D R

    2014-01-01

    Harmful algal blooms (HABs) expose aquatic organisms to multiple physical and chemical stressors during an acute time period. Algal toxins themselves may be altered by water chemistry parameters affecting their bioavailability and resultant toxicity. The purpose of this study was to determine the effects of two abiotic parameters (pH, inorganic metal salts) on the toxicity of fatty acid amides and fatty acids, two classes of lipids produced by harmful algae, including the golden alga, Prymnesium parvum, that are toxic to aquatic organisms. Rainbow trout gill cells were used as a model of the fish gill and exposed to single compounds and mixtures of compounds along with variations in pH level and concentration of inorganic metal salts. We employed artificial neural networks (ANNs) and standard ANOVA statistical analysis to examine and predict the effects of these abiotic parameters on the toxicity of fatty acid amides and fatty acids. Our results demonstrate that increasing pH levels increases the toxicity of fatty acid amides and inhibits the toxicity of fatty acids. This phenomenon is reversed at lower pH levels. Exposing gill cells to complex mixtures of chemical factors resulted in dramatic increases in toxicity compared to tests of single compounds for both the fatty acid amides and fatty acids. These findings highlight the potential of physicochemical factors to affect the toxicity of chemicals released during algal blooms and demonstrate drastic differences in the effect of pH on fatty acid amides and fatty acids. PMID:24240104

  1. Acidic pH resistance of grafted chitosan on dental implant.

    PubMed

    Campos, Doris M; Toury, Bérengère; D'Almeida, Mélanie; Attik, Ghania N; Ferrand, Alice; Renoud, Pauline; Grosgogeat, Brigitte

    2015-05-01

    Over the last decade, access to dental care has increasingly become a service requested by the population, especially in the case of dental implants. However, the major cause of implant failure is an inflammatory disease: peri-implantitis. Currently, the adhesion strength of antibacterial coatings at implant surfaces remains a problem to solve. In order to propose a functionalized implant with a resistant antibacterial coating, a novel method of chitosan immobilization at implant surface has been investigated. Functionalization of the pre-active titanium (Ti) surface was performed using triethoxysilylpropyl succinic anhydride (TESPSA) as a coupling agent which forms a stable double peptide bond with chitosan. The chitosan presence and the chemical resistibility of the coating under acid pH solutions (pH 5 and pH 3) were confirmed by FTIR-ATR and XPS analyses. Furthermore, peel test results showed high adhesive resistance of the TESPSA/chitosan coating at the substrate. Cytocompatibility was evaluated by cell morphology with confocal imaging. Images showed healthy morphology of human gingival fibroblasts (HGF-1). Finally, the reported method for chitosan immobilization on Ti surface via peptide bindings allows for the improvement of its adhesive capacities and resistibility while maintaining its cytocompatibility. Surface functionalization using the TESPSA/chitosan coupling method is noncytotoxic and stable even in drastic environments as found in oral cavity, thus making it a valuable candidate for clinical implantology applications. PMID:24972881

  2. Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion

    SciTech Connect

    Yu, I.-M.; Holdaway, H.A.; Chipman, P.R.; Kuhn, R.J.; Rossmann, M.G.; Chen, J.; Purdue

    2010-07-27

    Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.

  3. A pH dependent Raman and surface enhanced Raman spectroscopic studies of citrazinic acid aided by theoretical calculations

    NASA Astrophysics Data System (ADS)

    Sarkar, Sougata; Chowdhury, Joydeep; Dutta, Soumen; Pal, Tarasankar

    2016-12-01

    A pH dependent normal Raman scattering (NRS) and surface enhanced Raman scattering (SERS) spectral patterns of citrazinic acid (CZA), a biologically important molecule, have been investigated. The acid, with different pKa values (~ 4 and ~ 11) for the two different functional groups (-COOH and -OH groups), shows interesting range of color changes (yellow at pH ~ 14 and brown at pH ~ 2) with the variation in solution pH. Thus, depending upon the pH of the medium, CZA molecule can exist in various protonated and/or deprotonated forms. Here we have prescribed the existence different possible forms of CZA at different pH (Forms "C", "H" and "Dprot" at pH ~ 14 and Forms "A", "D", and "P" at pH ~ 2 respectively). The NRS spectra of these solutions and their respective SERS spectra over gold nanoparticles were recorded. The spectra clearly differ in their spectral profiles. For example the SERS spectra recorded with the CZA solution at pH ~ 2 shows blue shift for different bands compared to its NRS window e.g. 406 to 450 cm- 1, 616 to 632 cm- 1, 1332 to 1343 cm- 1 etc. Again, the most enhanced peak at ~ 1548 cm- 1 in NRS while in the SERS window this appears at ~ 1580 cm- 1. Similar observation was also made for CZA at pH ~ 14. For example, the 423 cm- 1 band in the NRS profile experience a blue shift and appears at ~ 447 cm- 1 in the SERS spectrum as well as other bands at ~ 850, ~ 1067 and ~ 1214 cm- 1 in the SERS window are markedly enhanced. It is also worth noting that the SERS spectra at the different pH also differ from each other. These spectral differences indicate the existence of various adsorptive forms of the CZA molecule depending upon the pH of the solution. Therefore based on the experimental findings we propose different possible molecular forms of CZA at different pH (acidic and alkaline) conditions. For example forms 'A', 'D' and 'P' existing in acidic pH (pH ~ 2) and three other deprotonated forms 'C', 'H' and 'Dprot' in alkaline pH (pH ~ 14). The DFT

  4. A pH dependent Raman and surface enhanced Raman spectroscopic studies of citrazinic acid aided by theoretical calculations.

    PubMed

    Sarkar, Sougata; Chowdhury, Joydeep; Dutta, Soumen; Pal, Tarasankar

    2016-12-01

    A pH dependent normal Raman scattering (NRS) and surface enhanced Raman scattering (SERS) spectral patterns of citrazinic acid (CZA), a biologically important molecule, have been investigated. The acid, with different pKa values (~4 and ~11) for the two different functional groups (-COOH and -OH groups), shows interesting range of color changes (yellow at pH~14 and brown at pH~2) with the variation in solution pH. Thus, depending upon the pH of the medium, CZA molecule can exist in various protonated and/or deprotonated forms. Here we have prescribed the existence different possible forms of CZA at different pH (Forms "C", "H" and "Dprot" at pH~14 and Forms "A", "D", and "P" at pH~2 respectively). The NRS spectra of these solutions and their respective SERS spectra over gold nanoparticles were recorded. The spectra clearly differ in their spectral profiles. For example the SERS spectra recorded with the CZA solution at pH~2 shows blue shift for different bands compared to its NRS window e.g. 406 to 450cm(-1), 616 to 632cm(-1), 1332 to 1343cm(-1) etc. Again, the most enhanced peak at ~1548cm(-1) in NRS while in the SERS window this appears at ~1580cm(-1). Similar observation was also made for CZA at pH~14. For example, the 423cm(-1) band in the NRS profile experience a blue shift and appears at ~447cm(-1) in the SERS spectrum as well as other bands at ~850, ~1067 and ~1214cm(-1) in the SERS window are markedly enhanced. It is also worth noting that the SERS spectra at the different pH also differ from each other. These spectral differences indicate the existence of various adsorptive forms of the CZA molecule depending upon the pH of the solution. Therefore based on the experimental findings we propose different possible molecular forms of CZA at different pH (acidic and alkaline) conditions. For example forms 'A', 'D' and 'P' existing in acidic pH (pH~2) and three other deprotonated forms 'C', 'H' and 'Dprot' in alkaline pH (pH~14). The DFT calculations for these

  5. Delivery of nucleic acids for cancer gene therapy: overcoming extra- and intra-cellular barriers.

    PubMed

    McErlean, Emma M; McCrudden, Cian M; McCarthy, Helen O

    2016-09-01

    The therapeutic potential of cancer gene therapy has been limited by the difficulty of delivering genetic material to target sites. Various biological and molecular barriers exist which need to be overcome before effective nonviral delivery systems can be applied successfully in oncology. Herein, various barriers are described and strategies to circumvent such obstacles are discussed, considering both the extracellular and intracellular setting. Development of multifunctional delivery systems holds much promise for the progression of gene delivery, and a growing body of evidence supports this approach involving rational design of vectors, with a unique molecular architecture. In addition, the potential application of composite gene delivery platforms is highlighted which may provide an alternative delivery strategy to traditional systemic administration. PMID:27582234

  6. d-Amino Acid Chemical Reporters Reveal Peptidoglycan Dynamics of an Intracellular Pathogen

    PubMed Central

    2012-01-01

    Peptidoglycan (PG) is an essential component of the bacterial cell wall. Although experiments with organisms in vitro have yielded a wealth of information on PG synthesis and maturation, it is unclear how these studies translate to bacteria replicating within host cells. We report a chemical approach for probing PG in vivo via metabolic labeling and bioorthogonal chemistry. A wide variety of bacterial species incorporated azide and alkyne-functionalized d-alanine into their cell walls, which we visualized by covalent reaction with click chemistry probes. The d-alanine analogues were specifically incorporated into nascent PG of the intracellular pathogen Listeria monocytogenes both in vitro and during macrophage infection. Metabolic incorporation of d-alanine derivatives and click chemistry detection constitute a facile, modular platform that facilitates unprecedented spatial and temporal resolution of PG dynamics in vivo. PMID:23240806

  7. Acidic pH retards the fibrillization of human islet amyloid polypeptide due to electrostatic repulsion of histidines

    NASA Astrophysics Data System (ADS)

    Li, Yang; Xu, Weixin; Mu, Yuguang; Zhang, John Z. H.

    2013-08-01

    The human Islet Amyloid Polypeptide (hIAPP) is the major constituent of amyloid deposits in pancreatic islets of type-II diabetes. IAPP is secreted together with insulin from the acidic secretory granules at a low pH of approximately 5.5 to the extracellular environment at a neutral pH. The increased accumulation of extracellular hIAPP in diabetes indicates that changes in pH may promote amyloid formation. To gain insights and underlying mechanisms of the pH effect on hIAPP fibrillogenesis, all-atom molecular dynamics simulations in explicit solvent model were performed to study the structural properties of five hIAPP protofibrillar oligomers, under acidic and neutral pH, respectively. In consistent with experimental findings, simulation results show that acidic pH is not conducive to the structural stability of these oligomers. This provides a direct evidence for a recent experiment [L. Khemtemourian, E. Domenech, J. P. F. Doux, M. C. Koorengevel, and J. A. Killian, J. Am. Chem. Soc. 133, 15598 (2011)], 10.1021/ja205007j, which suggests that acidic pH inhibits the fibril formation of hIAPP. In addition, a complementary coarse-grained simulation shows the repulsive electrostatic interactions among charged His18 residues slow down the dimerization process of hIAPP by twofold. Besides, our all-atom simulations reveal acidic pH mainly affects the local structure around residue His18 by destroying the surrounding hydrogen-bonding network, due to the repulsive interactions between protonated interchain His18 residues at acidic pH. It is also disclosed that the local interactions nearby His18 operating between adjacent β-strands trigger the structural transition, which gives hints to the experimental findings that the rate of hIAPP fibril formation and the morphologies of the fibrillar structures are strongly pH-dependent.

  8. Do pH and flavonoids influence hypochlorous acid-induced catalase inhibition and heme modification?

    PubMed

    Krych-Madej, Justyna; Gebicka, Lidia

    2015-09-01

    Hypochlorous acid (HOCl), highly reactive oxidizing and chlorinating species, is formed in the immune response to invading pathogens by the reaction of hydrogen peroxide with chloride catalyzed by the enzyme myeloperoxidase. Catalase, an important antioxidant enzyme, catalyzing decomposition of hydrogen peroxide to water and molecular oxygen, hampers in vitro HOCl formation, but is also one of the main targets for HOCl. In this work we have investigated HOCl-induced catalase inhibition at different pH, and the influence of flavonoids (catechin, epigallocatechin gallate and quercetin) on this process. It has been shown that HOCl-induced catalase inhibition is independent on pH in the range 6.0-7.4. Preincubation of catalase with epigallocatechin gallate and quercetin before HOCl treatment enhances the degree of catalase inhibition, whereas catechin does not affect this process. Our rapid kinetic measurements of absorption changes around the heme group have revealed that heme modification by HOCl is mainly due to secondary, intramolecular processes. The presence of flavonoids, which reduce active catalase intermediate, Compound I to inactive Compound II have not influenced the kinetics of HOCl-induced heme modification. Possible mechanisms of the reaction of hypochlorous acid with catalase are proposed and the biological consequences are discussed. PMID:26116387

  9. Ultrasonic absorption in aqueous solutions of amino acids at neutral pH

    NASA Astrophysics Data System (ADS)

    Nishikawa, S.; Ohno, T.; Huang, H.; Yoshizuka, K.; Jordan, F.

    2003-05-01

    Ultrasonic absorption coefficients in aqueous solutions of glycine, L-alanine, imidazole, L-phenylalanine, L-histidine and L-tryptophan at neutral pH were measured in the range from 0.8 to 220 MHz at 25 °C. A characteristic ultrasonic relaxation phenomenon was observed only in the solution of L-histidine with a relaxation frequency at around 2 MHz at neutral pH. It was proposed from the concentration independent relaxation frequency and the linear concentration dependence of the maximum absorption per wavelength that the relaxation mechanism was associated with a perturbation of the rotational isomeric equilibrium of the L-histidine molecule. The existence of two rotational isomeric forms of L-histidine in water was examined by semiempirical quantum chemical methods, in order to determine the free energy difference between the two states. The forward and backward rate constants were determined from the relaxation frequency and the energy change. Also, the standard volume change of the reaction was estimated from the concentration dependence of the maximum absorption per wavelength. It was speculated that L-histidine fulfills a specific function among amino acids because of the rotational motion in the molecule, in addition to its well-established acid-base properties.

  10. Elaidate, an 18-Carbon Trans-monoenoic Fatty Acid, but not Physiological Fatty Acids Increases Intracellular Zn2+ in Human Macrophages

    PubMed Central

    Zacherl, Janelle R.; Tourkova, Irina; St Croix, Claudette M.; Robinson, Lisa J.; Peck Palmer, Octavia M.; Mihalik, Stephanie J.; Blair, Harry C.

    2015-01-01

    Artificial trans fatty acids promote atherosclerosis by blocking macrophage clearance of cell debris. Classical fatty-acid response mechanisms include TLR4-NF-κB activation, and Erk1/2 phosphorylation, but these may not indicate long-term mechanisms. Indeed, nuclear NF-κB was increased by 60 minute treatment by 30 μM of the 18 carbon trans unsaturated fatty acid elaidic acid (elaidate), the physiological cis-unsaturated fatty acid oleic acid (oleate), and the 18 or 16 carbon saturated fatty acids stearic and palmitic acid (stearate or palmitate). However, except for stearate, effects on related pathways were minimal at 44 hours. To determine longer term effects of trans fatty acids, we compared whole exome mRNA expression of (trans) elaidate to (cis) oleate, 30 μM, at 44 hours in human macrophages. We found that elaidate changed Zn2+-homeostasis gene mRNAs markedly. This might be important because Zn2+ is a major regulator of macrophage activity. Messenger RNAs of seven Zn2+-binding metallothioneins decreased 2–4 fold; the zinc importer SLC39A10 increased 2-fold, in elaidate relative to oleate-treated cells. Results were followed by quantitative PCR comparing cis, trans, and saturated fatty acid effects on Zn2+-homeostasis gene mRNAs. This confirmed that elaidate uniquely decreased metallothionein expression and increased SLC39A10 at 44 hours. Further, intracellular Zn2+ was measured using N-(carboxymethyl)-N-[2-[2-[2(carboxymethyl)amino]-5-(2,7,-difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)-phenoxy]-ethoxy]-4-methoxyphenyl]glycine, acetoxymethyl ester (FluoZin-3-AM). This showed that, at 44 hours, only cells treated with elaidate had increased Zn2+. The durable effect of elaidate on Zn2+ activation is a novel and specific effect of trans fatty acids on peripheral macrophage metabolism. PMID:25358453

  11. Microbial sulfate reduction and metal attenuation in pH 4 acid mine water

    USGS Publications Warehouse

    Church, C.D.; Wilkin, R.T.; Alpers, C.N.; Rye, R.O.; Blaine, R.B.

    2007-01-01

    Sediments recovered from the flooded mine workings of the Penn Mine, a Cu-Zn mine abandoned since the early 1960s, were cultured for anaerobic bacteria over a range of pH (4.0 to 7.5). The molecular biology of sediments and cultures was studied to determine whether sulfate-reducing bacteria (SRB) were active in moderately acidic conditions present in the underground mine workings. Here we document multiple, independent analyses and show evidence that sulfate reduction and associated metal attenuation are occurring in the pH-4 mine environment. Water-chemistry analyses of the mine water reveal: (1) preferential complexation and precipitation by H2S of Cu and Cd, relative to Zn; (2) stable isotope ratios of 34S/32S and 18O/16O in dissolved SO4 that are 2-3 ??? heavier in the mine water, relative to those in surface waters; (3) reduction/oxidation conditions and dissolved gas concentrations consistent with conditions to support anaerobic processes such as sulfate reduction. Scanning electron microscope (SEM) analyses of sediment show 1.5-micrometer, spherical ZnS precipitates. Phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analyses of Penn Mine sediment show a high biomass level with a moderately diverse community structure composed primarily of iron- and sulfate-reducing bacteria. Cultures of sediment from the mine produced dissolved sulfide at pH values near 7 and near 4, forming precipitates of either iron sulfide or elemental sulfur. DGGE coupled with sequence and phylogenetic analysis of 16S rDNA gene segments showed populations of Desulfosporosinus and Desulfitobacterium in Penn Mine sediment and laboratory cultures. ?? 2007 Church et al; licensee BioMed Central Ltd.

  12. Microbial sulfate reduction and metal attenuation in pH 4 acid mine water

    PubMed Central

    Church, Clinton D; Wilkin, Richard T; Alpers, Charles N; Rye, Robert O; McCleskey, R Blaine

    2007-01-01

    Sediments recovered from the flooded mine workings of the Penn Mine, a Cu-Zn mine abandoned since the early 1960s, were cultured for anaerobic bacteria over a range of pH (4.0 to 7.5). The molecular biology of sediments and cultures was studied to determine whether sulfate-reducing bacteria (SRB) were active in moderately acidic conditions present in the underground mine workings. Here we document multiple, independent analyses and show evidence that sulfate reduction and associated metal attenuation are occurring in the pH-4 mine environment. Water-chemistry analyses of the mine water reveal: (1) preferential complexation and precipitation by H2S of Cu and Cd, relative to Zn; (2) stable isotope ratios of 34S/32S and 18O/16O in dissolved SO4 that are 2–3 ‰ heavier in the mine water, relative to those in surface waters; (3) reduction/oxidation conditions and dissolved gas concentrations consistent with conditions to support anaerobic processes such as sulfate reduction. Scanning electron microscope (SEM) analyses of sediment show 1.5-micrometer, spherical ZnS precipitates. Phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analyses of Penn Mine sediment show a high biomass level with a moderately diverse community structure composed primarily of iron- and sulfate-reducing bacteria. Cultures of sediment from the mine produced dissolved sulfide at pH values near 7 and near 4, forming precipitates of either iron sulfide or elemental sulfur. DGGE coupled with sequence and phylogenetic analysis of 16S rDNA gene segments showed populations of Desulfosporosinus and Desulfitobacterium in Penn Mine sediment and laboratory cultures. PMID:17956615

  13. Microbial sulfate reduction and metal attenuation in pH 4 acid mine water.

    PubMed

    Church, Clinton D; Wilkin, Richard T; Alpers, Charles N; Rye, Robert O; McCleskey, R Blaine

    2007-01-01

    Sediments recovered from the flooded mine workings of the Penn Mine, a Cu-Zn mine abandoned since the early 1960s, were cultured for anaerobic bacteria over a range of pH (4.0 to 7.5). The molecular biology of sediments and cultures was studied to determine whether sulfate-reducing bacteria (SRB) were active in moderately acidic conditions present in the underground mine workings. Here we document multiple, independent analyses and show evidence that sulfate reduction and associated metal attenuation are occurring in the pH-4 mine environment. Water-chemistry analyses of the mine water reveal: (1) preferential complexation and precipitation by H2S of Cu and Cd, relative to Zn; (2) stable isotope ratios of 34S/32S and 18O/16O in dissolved SO4 that are 2-3 per thousand heavier in the mine water, relative to those in surface waters; (3) reduction/oxidation conditions and dissolved gas concentrations consistent with conditions to support anaerobic processes such as sulfate reduction. Scanning electron microscope (SEM) analyses of sediment show 1.5-micrometer, spherical ZnS precipitates. Phospholipid fatty acid (PLFA) and denaturing gradient gel electrophoresis (DGGE) analyses of Penn Mine sediment show a high biomass level with a moderately diverse community structure composed primarily of iron- and sulfate-reducing bacteria. Cultures of sediment from the mine produced dissolved sulfide at pH values near 7 and near 4, forming precipitates of either iron sulfide or elemental sulfur. DGGE coupled with sequence and phylogenetic analysis of 16S rDNA gene segments showed populations of Desulfosporosinus and Desulfitobacterium in Penn Mine sediment and laboratory cultures. PMID:17956615

  14. pH Titratable Superparamagnetic Iron Oxide for Improved Nanoparticle Accumulation in Acidic Tumor Microenvironments

    PubMed Central

    Crayton, Samuel H.; Tsourkas, Andrew

    2011-01-01

    A wide variety of nanoparticle platforms are being developed for the diagnosis and treatment of malignancy. While many of these are passively targeted or rely on receptor-ligand interactions, metabolically directed nanoparticles provide a complementary approach. It is known that both primary and secondary events in tumorigensis alter the metabolic profile of developing and metastatic cancers. One highly conserved metabolic phenotype is a state of up-regulated glycolysis and reduced use of oxidative phosphorylation, even when oxygen tension is not limiting. This metabolic shift, termed the Warburg effect, creates a “hostile” tumor microenvironment with increased levels of lactic acid and low extracellular pH. In order to exploit this phenomenon and improve the delivery of nanoparticle platforms to a wide variety of tumors, a pH-responsive iron oxide nanoparticle was designed. Specifically, glycol chitosan (GC), a water-soluble polymer with pH titratable charge, was conjugated to the surface of superparamagnetic iron oxide nanoparticles (SPIO) to generate a T2*-weighted MR contrast agent that responds to alterations in its surrounding pH. Compared to control nanoparticles that lack pH sensitivity, these GC-SPIO nanoparticles demonstrated potent pH-dependent cellular association and MR contrast in vitro. In murine tumor models GC-SPIO also generated robust T2*-weighted contrast, which correlated with increased delivery of the agent to the tumor site, measured quantitatively by inductively coupled plasma mass spectrometry. Importantly, the increased delivery of GC-SPIO nanoparticles cannot be solely attributed to the commonly observed enhanced permeability and retention effect, since these nanoparticles have similar physical properties and blood circulation times as control agents. PMID:22035454

  15. Predicting Thermodynamic Behaviors of Non-Protein Amino Acids as a Function of Temperature and pH

    NASA Astrophysics Data System (ADS)

    Kitadai, Norio

    2016-03-01

    Why does life use α-amino acids exclusively as building blocks of proteins? To address that fundamental question from an energetic perspective, this study estimated the standard molal thermodynamic data for three non-α-amino acids (β-alanine, γ-aminobutyric acid, and ɛ-aminocaproic acid) and α-amino- n-butyric acid in their zwitterionic, negative, and positive ionization states based on the corresponding experimental measurements reported in the literature. Temperature dependences of their heat capacities were described based on the revised Helgeson-Kirkham-Flowers (HKF) equations of state. The obtained dataset was then used to calculate the standard molal Gibbs energies ( ∆G o) of the non-α-amino acids as a function of temperature and pH. Comparison of their ∆G o values with those of α-amino acids having the same molecular formula showed that the non-α-amino acids have similar ∆G o values to the corresponding α-amino acids in physiologically relevant conditions (neutral pH, <100 °C). In acidic and alkaline pH, the non-α-amino acids are thermodynamically more stable than the corresponding α-ones over a broad temperature range. These results suggest that the energetic cost of synthesis is not an important selection pressure to incorporate α-amino acids into biological systems.

  16. [Effects of simulated acid rain on respiration rate of cropland system with different soil pH].

    PubMed

    Zhu, Xue-zhu; Zhang, Gao-chuan; Li, Hui

    2009-10-15

    To evaluate the effects of acid rain on the respiration rate of cropland system, an outdoor pot experiment was conducted with paddy soils of pH 5.48 (S1), pH 6.70 (S1) and pH 8.18 (S3) during the 2005-2007 wheat-growing seasons. The cropland system was exposed to acid rain by spraying the wheat foliage and irrigating the soil with simulated rainwater of T1 (pH 6.0), T2 (pH 6.0, ionic concentration was twice as rainwater T1), and T3 (pH 4.4, ionic concentration was twice as rainwater T1), respectively. The static opaque chamber-gas chromatograph method was used to measure CO2 fluxes from cropland system. The results showed that acid rain affected the respiration rate of cropland system through crop plant, and the cropland system could adapt to acid rain. Acid rainwater significantly increased the average respiration rate in alkaline soil (S3) cropland system, while it had no significant effects on the average respiration rate in neutral soil (S2) and acidic soil (S1) cropland systems. During 2005-2006, after the alkaline soil cropland system was treated with rainwater T3, the average respiration rate was 23.6% and 27.6% higher than that of alkaline soil cropland system treated with rainwater T1 and T2, respectively. During March to April, the respiration rate was enhanced with the increase of rainwater ionic concentration, while it was dropped with the decrease of rainwater pH value in acidic soil cropland system. It was demonstrated that soil pH and crop plant played important roles on the respiration rate of cropland system. PMID:19968099

  17. Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein.

    PubMed Central

    Hanakam, F; Albrecht, R; Eckerskorn, C; Matzner, M; Gerisch, G

    1996-01-01

    Hisactophilins are myristoylated proteins that are rich in histidine residues and known to exist in Dictyostelium cells in a plasma membrane-bound and a soluble cytoplasmic state. Intracellular translocation of these proteins in response to pH changes was monitored using hisactophilin fusions with green fluorescent protein (GFP) and confocal laser scanning microscopy. Both the normal and a mutated non-myristoylated fusion protein shuffled within the cells in a pH-dependent manner. After lowering the pH, these proteins translocated within minutes between the cytoplasm, the plasma membrane and the nucleus. The role of histidine clusters on the surface of hisactophilin molecules in binding of the proteins to the plasma membrane and in their transfer to the nucleus is discussed on the basis of a pH switch mechanism. Images PMID:8670794

  18. Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells

    PubMed Central

    Li, Dan

    2014-01-01

    Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca2+ in FLO cells, an increase that is blocked by Ca2+-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca2+ increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA. PMID:24699332

  19. Enzymatic characterization of peptidic materials isolated from aqueous solutions of ammonium cyanide (pH 9) and hydrocyanic acid (pH 6) exposed to ionizing radiation.

    PubMed

    Niketic, V; Draganić, Z; Nesković, S; Draganić, I

    1982-01-01

    The enzymatic digestion of some radiolytically produced peptidic materials was examined. The substrates were compounds isolated from 0.1 molar solutions of NH4CN (pH 9) and HCN (pH 6), after their exposure to gamma rays from a 60Co source (15-20 Mrad doses). Commercial proteolytic enzymes pronase and aminopeptidase M were used. The examined materials were of composite nature and proteolytic action was systematically observed after their subsequent purification. In some fractions the effect was found to be positive with up to 30% of peptide bonds cleaved with respect to the amino acid content. These findings support our previous conclusions on the free radical induced formation of peptidic backbones without the intervention of amino acids. Some side effects were also noted which might be of interest in observations on enzymatic cleavage of other composite peptidic materials of abiotic origin. PMID:6124639

  20. Sensitive detection of strong acidic condition by a novel rhodamine-based fluorescent pH chemosensor.

    PubMed

    Tan, Jia-Lian; Yang, Ting-Ting; Liu, Yu; Zhang, Xue; Cheng, Shu-Jin; Zuo, Hua; He, Huawei

    2016-05-01

    A novel rhodamine-based fluorescent pH probe responding to extremely low pH values has been synthesized and characterized. This probe showed an excellent photophysical response to pH on the basis that the colorless spirocyclic structure under basic conditions opened to a colored and highly fluorescent form under extreme acidity. The quantitative relationship between fluorescence intensity and pH value (1.75-2.62) was consistent with the equilibrium equation pH = pKa + log[(Imax - I)/(I - Imin )]. This sensitive pH probe was also characterized with good reversibility and no interaction with interfering metal ions, and was successfully applied to image Escherichia coli under strong acidity. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26467547

  1. Intracellular traffic of the lysine and glutamic acid rich protein KERP1 reveals features of endomembrane organization in Entamoeba histolytica.

    PubMed

    Perdomo, Doranda; Manich, Maria; Syan, Sylvie; Olivo-Marin, Jean-Christophe; Dufour, Alexandre C; Guillén, Nancy

    2016-08-01

    The development of amoebiasis is influenced by the expression of the lysine and glutamic acid rich protein 1 (KERP1), a virulence factor involved in Entamoeba histolytica adherence to human cells. Up to date, it is unknown how the protein transits the parasite cytoplasm towards the plasma membrane, specially because this organism lacks a well-defined endoplasmic reticulum (ER) and Golgi apparatus. In this work we demonstrate that KERP1 is present at the cell surface and in intracellular vesicles which traffic in a pathway that is independent of the ER-Golgi anterograde transport. The intracellular displacement of vesicles enriched in KERP1 relies on the actin-rich cytoskeleton activities. KERP1 is also present in externalized vesicles deposited on the surface of human cells. We further report the interactome of KERP1 with its association to endomembrane components and lipids. The model for KERP1 traffic here proposed hints for the first time elements of the endocytic and exocytic paths of E. histolytica. PMID:26857352

  2. Intracellular Self-Assembly of Cyclic d-Luciferin Nanoparticles for Persistent Bioluminescence Imaging of Fatty Acid Amide Hydrolase.

    PubMed

    Yuan, Yue; Wang, Fuqiang; Tang, Wei; Ding, Zhanling; Wang, Lin; Liang, Lili; Zheng, Zhen; Zhang, Huafeng; Liang, Gaolin

    2016-07-26

    Fatty acid amide hydrolase (FAAH) overexpression induces several disorder symptoms in nerve systems, and therefore long-term tracing of FAAH activity in vivo is of high importance but remains challenging. Current bioluminescence (BL) methods are limited in detecting FAAH activity within 5 h. Herein, by rational design of a latent BL probe (d-Cys-Lys-CBT)2 (1), we developed a "smart" method of intracellular reduction-controlled self-assembly and FAAH-directed disassembly of its cyclic d-luciferin-based nanoparticles (i.e., 1-NPs) for persistent BL imaging of FAAH activity in vitro, in cells, and in vivo. Using aminoluciferin methyl amide (AMA), Lys-amino-d-luciferin (Lys-Luc), and amino-d-luciferin (NH2-Luc) as control BL probes, we validated that the persistent BL of 1 from luciferase-expressing cells or tumors was controlled by the activity of intracellular FAAH. With the property of long-term tracing of FAAH activity in vivo of 1, we envision that our BL precursor 1 could probably be applied for in vivo screening of FAAH inhibitors and the diagnosis of their related diseases (or disorders) in the future. PMID:27348334

  3. Lower pH values of weakly acidic refluxes as determinants of heartburn perception in gastroesophageal reflux disease patients with normal esophageal acid exposure.

    PubMed

    de Bortoli, N; Martinucci, I; Savarino, E; Franchi, R; Bertani, L; Russo, S; Ceccarelli, L; Costa, F; Bellini, M; Blandizzi, C; Savarino, V; Marchi, S

    2016-01-01

    Multichannel impedance pH monitoring has shown that weakly acidic refluxes are able to generate heartburn. However, data on the role of different pH values, ranging between 4 and 7, in the generation of them are lacking. The aim of this study was to evaluate whether different pH values of weakly acidic refluxes play a differential role in provoking reflux symptoms in endoscopy-negative patients with physiological esophageal acid exposure time and positive symptom index and symptom association probability for weakly acidic refluxes. One hundred and forty-three consecutive patients with gastroesophageal reflux disease, nonresponders to proton pump inhibitors (PPIs), were allowed a washout from PPIs before undergoing: upper endoscopy, esophageal manometry, and multichannel impedance pH monitoring. In patients with both symptom index and symptom association probability positive for weakly acidic reflux, each weakly acidic reflux was evaluated considering exact pH value, extension, physical characteristics, and correlation with heartburn. Forty-five patients with normal acid exposure time and positive symptom association probability for weakly acidic reflux were identified. The number of refluxes not heartburn related was higher than those heartburn related. In all distal and proximal liquid refluxes, as well as in distal mixed refluxes, the mean pH value of reflux events associated with heartburn was significantly lower than that not associated. This condition was not confirmed for proximal mixed refluxes. Overall, a low pH of weakly acidic reflux represents a determinant factor in provoking heartburn. This observation contributes to better understand the pathophysiology of symptoms generated by weakly acidic refluxes, paving the way toward the search for different therapeutic approaches to this peculiar condition of esophageal hypersensitivity. PMID:25212408

  4. Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

    PubMed Central

    2010-01-01

    Background Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. Results Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. Conclusions 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary

  5. Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

    PubMed

    Bellotti, Marta; Salis, Annalisa; Grozio, Alessia; Damonte, Gianluca; Vigliarolo, Tiziana; Galatini, Andrea; Zocchi, Elena; Benatti, Umberto; Millo, Enrico

    2015-01-01

    The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds. PMID:25496807

  6. The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages.

    PubMed

    Cumley, Nicola J; Smith, Leanne M; Anthony, Mark; May, Robin C

    2012-05-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  7. The CovS/CovR Acid Response Regulator Is Required for Intracellular Survival of Group B Streptococcus in Macrophages

    PubMed Central

    Cumley, Nicola J.; Smith, Leanne M.; Anthony, Mark

    2012-01-01

    Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome. PMID:22331428

  8. Intracellular location of rabbit poxvirus nucleic acid within infected cells as determined by in situ hybridization.

    PubMed Central

    Minnigan, H; Moyer, R W

    1985-01-01

    The intracellular location of rabbit poxvirus DNA within cells during the course of infection has been determined by the hybridization in situ of labeled viral DNA probes to uninfected and infected cells under various conditions. Extensive control experiments were performed to demonstrate that DNA could be detected selectively and accurately within the cell. Our results suggest that rabbit poxvirus DNA is located only within the cytoplasm during the reproductive cycle, and we found no evidence that viral DNA enters the cell nucleus. The pattern of hybridization of viral DNA at early times (1 and 2 h postinfection) and in the presence of inhibitors of viral DNA synthesis suggests that there may be an association between the input viral DNA and some structural component of the host cell. A number of observations support the hypothesis that the host cell nucleus is required for a productive poxvirus infection. Our results are discussed in terms of the possible role of the nucleus in the replication of poxviruses. Images PMID:2991586

  9. Effect of pH alkaline salts of fatty acids on the inhibition of bacteria associated with poultry processing

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The agar diffusion assay was used to examine the effect of pH on the ability of alkaline salts of three fatty acids (FA) to inhibit growth of bacteria associated with poultry processing. FA solutions were prepared by dissolving 0.5 M concentrations of caprylic, capric, or lauric acid in separate ali...

  10. Ruminant Nutrition Symposium: Role of fermentation acid absorption in the regulation of ruminal pH.

    PubMed

    Aschenbach, J R; Penner, G B; Stumpff, F; Gäbel, G

    2011-04-01

    Highly fermentable diets are rapidly converted to organic acids [i.e., short-chain fatty acids (SCFA) and lactic acid] within the rumen. The resulting release of protons can constitute a challenge to the ruminal ecosystem and animal health. Health disturbances, resulting from acidogenic diets, are classified as subacute and acute acidosis based on the degree of ruminal pH depression. Although increased acid production is a nutritionally desired effect of increased concentrate feeding, the accumulation of protons in the rumen is not. Consequently, mechanisms of proton removal and their quantitative importance are of major interest. Saliva buffers (i.e., bicarbonate, phosphate) have long been identified as important mechanisms for ruminal proton removal. An even larger proportion of protons appears to be removed from the rumen by SCFA absorption across the ruminal epithelium, making efficiency of SCFA absorption a key determinant for the individual susceptibility to subacute ruminal acidosis. Proceeding initially from a model of exclusively diffusional absorption of fermentation acids, several protein-dependent mechanisms have been discovered over the last 2 decades. Although the molecular identity of these proteins is mostly uncertain, apical acetate absorption is mediated, to a major degree, via acetate-bicarbonate exchange in addition to another nitrate-sensitive, bicarbonate-independent transport mechanism and lipophilic diffusion. Propionate and butyrate also show partially bicarbonate-dependent transport modes. Basolateral efflux of SCFA and their metabolites has to be mediated primarily by proteins and probably involves the monocarboxylate transporter (MCT1) and anion channels. Although the ruminal epithelium removes a large fraction of protons from the rumen, it also recycles protons to the rumen via apical sodium-proton exchanger, NHE. The latter is stimulated by ruminal SCFA absorption and salivary Na(+) secretion and protects epithelial integrity. Finally

  11. Fatty acid-binding proteins (FABPs) are intracellular carriers for Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD).

    PubMed

    Elmes, Matthew W; Kaczocha, Martin; Berger, William T; Leung, KwanNok; Ralph, Brian P; Wang, Liqun; Sweeney, Joseph M; Miyauchi, Jeremy T; Tsirka, Stella E; Ojima, Iwao; Deutsch, Dale G

    2015-04-01

    Δ(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. PMID:25666611

  12. Fatty Acid-binding Proteins (FABPs) Are Intracellular Carriers for Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD)*

    PubMed Central

    Elmes, Matthew W.; Kaczocha, Martin; Berger, William T.; Leung, KwanNok; Ralph, Brian P.; Wang, Liqun; Sweeney, Joseph M.; Miyauchi, Jeremy T.; Tsirka, Stella E.; Ojima, Iwao; Deutsch, Dale G.

    2015-01-01

    Δ9-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders. PMID:25666611

  13. Nonenzymatic browning reaction of essential amino acids: effect of pH on caramelization and Maillard reaction kinetics.

    PubMed

    Ajandouz, E H; Puigserver, A

    1999-05-01

    The interaction between glucose and essential amino acids at 100 degrees C at pH values ranging from 4.0 to 12.0 was investigated by monitoring the disappearance of glucose and amino acids as well as the appearance of brown color. Lysine was the most strongly destroyed amino acid, followed by threonine which induced very little additional browning as compared with that undergone by glucose. Around neutrality, the nonenzymatic browning followed pseudo-zero-order kinetics after a lag time, while the glucose and amino acid losses did not follow first-order kinetics at any of the pH values tested. Glucose was more strongly destroyed than all of the essential amino acids, the losses of which are really small at pH values lower than 9.0. However, glucose was less susceptible to thermal degradation in the presence of amino acids, especially at pH 8.0 with threonine and at pH 10.0 with lysine. The contribution of the caramelization reaction to the overall nonenzymatic browning above neutrality should lead to an overestimation of the Maillard reaction in foods. PMID:10552453

  14. Intracellular self-assembly based multi-labeling of key viral components: Envelope, capsid and nucleic acids.

    PubMed

    Wen, Li; Lin, Yi; Zhang, Zhi-Ling; Lu, Wen; Lv, Cheng; Chen, Zhi-Liang; Wang, Han-Zhong; Pang, Dai-Wen

    2016-08-01

    Envelope, capsid and nucleic acids are key viral components that are all involved in crucial events during virus infection. Thus simultaneous labeling of these key components is an indispensable prerequisite for monitoring comprehensive virus infection process and dissecting virus infection mechanism. Baculovirus was genetically tagged with biotin on its envelope protein GP64 and enhanced green fluorescent protein (EGFP) on its capsid protein VP39. Spodoptera frugiperda 9 (Sf9) cells were infected by the recombinant baculovirus and subsequently fed with streptavidin-conjugated quantum dots (SA-QDs) and cell-permeable nucleic acids dye SYTO 82. Just by genetic engineering and virus propagation, multi-labeling of envelope, capsid and nucleic acids was spontaneously accomplished during virus inherent self-assembly process, significantly simplifying the labeling process while maintaining virus infectivity. Intracellular dissociation and transportation of all the key viral components, which was barely reported previously, was real-time monitored based on the multi-labeling approach, offering opportunities for deeply understanding virus infection and developing anti-virus treatment. PMID:27209260

  15. Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase

    SciTech Connect

    Fujita, Akiko; Sato, Chihiro; Kitajima, Ken. E-mail: kitajima@agr.nagoya-u.ac.jp

    2007-03-30

    The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences.

  16. The liver fatty acid binding protein--comparison of cavity properties of intracellular lipid-binding proteins.

    PubMed

    Thompson, J; Ory, J; Reese-Wagoner, A; Banaszak, L

    1999-02-01

    The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common beta-barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited 'portal' region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound. PMID:10331654

  17. Annexins V and XII insert into bilayers at mildly acidic pH and form ion channels.

    PubMed

    Isas, J M; Cartailler, J P; Sokolov, Y; Patel, D R; Langen, R; Luecke, H; Hall, J E; Haigler, H T

    2000-03-21

    The functional hallmark of annexins is the ability to bind to the surface of phospholipid membranes in a reversible, Ca(2+)-dependent manner. We now report that human annexin V and hydra annexin XII reversibly bound to phospholipid vesicles in the absence of Ca(2+) at low pH; half-maximal vesicle association occurred at pH 5.3 and 5. 8, respectively. The following biochemical data support the hypothesis that these annexins insert into bilayers at mildly acidic pH. First, a photoactivatable reagent (3-trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine) which selectively labels proteins exposed to the hydrophobic domain of bilayers reacted with these annexins at pH 5.0 and below but not at neutral pH. Second, in a Triton X-114 partitioning assay, annexins V and XII act as integral membrane proteins at low pH and as hydrophilic proteins at neutral pH; in the presence of phospholipids half-maximal partitioning into detergent occurred at pH approximately 5.0. Finally, annexin V or XII formed single channels in phospholipid bilayers at low pH but not at neutral pH. A model is discussed in which the concentrations of H(+) and Ca(2+) regulate the reversible conversion of three forms of annexins-soluble, peripheral membrane, and transmembrane. PMID:10715122

  18. Aphid amino acid transporter regulates glutamine supply to intracellular bacterial symbionts.

    PubMed

    Price, Daniel R G; Feng, Honglin; Baker, James D; Bavan, Selvan; Luetje, Charles W; Wilson, Alex C C

    2014-01-01

    Endosymbiotic associations have played a major role in evolution. However, the molecular basis for the biochemical interdependence of these associations remains poorly understood. The aphid-Buchnera endosymbiosis provides a powerful system to elucidate how these symbioses are regulated. In aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium Buchnera aphidicola. Buchnera cells are densely packed in specialized aphid bacteriocyte cells. Here we confirm that five putative amino acid transporters are highly expressed and/or highly enriched in Acyrthosiphon pisum bacteriocyte tissues. When expressed in Xenopus laevis oocytes, two bacteriocyte amino acid transporters displayed significant levels of glutamine uptake, with transporter ACYPI001018, LOC100159667 (named here as Acyrthosiphon pisum glutamine transporter 1, ApGLNT1) functioning as the most active glutamine transporter. Transporter ApGLNT1 has narrow substrate selectivity, with high glutamine and low arginine transport capacity. Notably, ApGLNT1 has high binding affinity for arginine, and arginine acts as a competitive inhibitor for glutamine transport. Using immunocytochemistry, we show that ApGLNT1 is localized predominantly to the bacteriocyte plasma membrane, a location consistent with the transport of glutamine from A. pisum hemolymph to the bacteriocyte cytoplasm. On the basis of functional transport data and localization, we propose a substrate feedback inhibition model in which the accumulation of the essential amino acid arginine in A. pisum hemolymph reduces the transport of the precursor glutamine into bacteriocytes, thereby regulating amino acid biosynthesis in the bacteriocyte. Structural similarities in the arrangement of hosts and symbionts across endosymbiotic systems suggest that substrate feedback inhibition may be mechanistically important in other endosymbioses. PMID:24367072

  19. Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes

    SciTech Connect

    Frost, S.J.; Raja, R.H.; Weigel, P.H. )

    1990-11-13

    125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4{degrees}C increased greater than 10-fold at pH 5.0 as compared to pH 7.

  20. Intracellular potassium as a possible inducer of amino acid transport across hamster jejunal enterocytes.

    PubMed Central

    Cremaschi, D; James, P S; Meyer, G; Rossetti, C; Smith, M W

    1986-01-01

    Brush border membrane potentials (Vm), intracellular K+ activity (aiK) and alanine uptake were measured in different parts of villi in mid-jejunal tissue taken from hamsters fed different amounts of food at high and low environmental temperatures. Basal villus enterocytes (Y cells) were found to have lower values for Vm and aiK than upper villus enterocytes (O cells). Alanine uptake was confined to O cells. The position on the villus where values for Vm and aiK changed, and where alanine uptake could first be seen to take place, depended on the energy intake and environmental temperature at which hamsters were kept. Na+-dependent alanine uptake and Vm were both higher in O cells of hamsters fed 10 kcal day-1 at 30 degrees C (10 k/30 degrees C) compared with animals fed 30 kcal day-1 at an environmental temperature of 12 degrees C (30 k/12 degrees C hamsters). The rates at which enterocytes migrated along the crypt-villus axis, measured separately in thymidine-labelling experiments, were 6.9 and 16.1 micron h-1 for 10 k/30 degrees C and 30 k/12 degrees C hamsters respectively. Both Vm and aiK were estimated, from these measurements, to have increased significantly by the time enterocytes became 30 h old. Alanine uptake began 15 h later. Neither of these parameters were influenced by previous adaptation conditions. The close temporal and variable positional relationship found between changes in aiK and onset of transport suggests that early changes in electrolyte composition might initiate a second phase of development enabling the enterocyte to absorb nutrients. The possibility that other ions besides K+ might also be involved in this aspect of regulation is also discussed. PMID:3795055

  1. Acidic amino acids in the first intracellular loop contribute to voltage- and calcium- dependent gating of anoctamin1/TMEM16A.

    PubMed

    Xiao, Qinghuan; Cui, Yuanyuan

    2014-01-01

    Anoctamin1 (Ano1, or TMEM16A) is a Ca2+-activated chloride channel that is gated by both voltage and Ca2+. We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τfast) that represented voltage-dependent mode, and a slow component (τslow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τfast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the τslow, and produced a 2-3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1. PMID:24901998

  2. Acidic intracellular Ca(2+) stores and caveolae in Ca(2+) signaling and diabetes.

    PubMed

    Guerrero-Hernandez, Agustin; Gallegos-Gomez, Martin Leonardo; Sanchez-Vazquez, Victor Hugo; Lopez-Mendez, Maria Cristina

    2014-11-01

    Acidic Ca(2+) stores, particularly lysosomes, are newly discovered players in the well-orchestrated arena of Ca(2+) signaling and we are at the verge of understanding how lysosomes accumulate Ca(2+) and how they release it in response to different chemical, such as NAADP, and physical signals. Additionally, it is now clear that lysosomes play a key role in autophagy, a process that allows cells to recycle components or to eliminate damaged structures to ensure cellular well-being. Moreover, lysosomes are being unraveled as hubs that coordinate both anabolism via insulin signaling and catabolism via AMPK. These acidic vesicles have close contact with the ER and there is a bidirectional movement of information between these two organelles that exquisitely regulates cell survival. Lysosomes also connect with plasma membrane where caveolae are located as specialized regions involved in Ca(2+) and insulin signaling. Alterations of all these signaling pathways are at the core of insulin resistance and diabetes. PMID:25182518

  3. Acidic pH and short-chain fatty acids activate Na+ transport but differentially modulate expression of Na+/H+ exchanger isoforms 1, 2, and 3 in omasal epithelium.

    PubMed

    Lu, Zhongyan; Yao, Lei; Jiang, Zhengqian; Aschenbach, Jörg R; Martens, Holger; Shen, Zanming

    2016-01-01

    activate Na(+) transport but suppress the expression of NHE2 and NHE3 in the longer term. By contrast, the expression of NHE1 is increased by SCFA and acidic pH, indicating a prominent role for NHE1 in the regulation of intracellular pH of omasal epithelium. Our results suggest a regulatable Na(+) absorption in ruminal and omasal epithelium. It is of benefit for intracellular pH homeostasis and highly relevant to dairy cows fed on high-concentrate diets. PMID:26547645

  4. Hollow spherical nucleic acids for intracellular gene regulation based upon biocompatible silica shells.

    PubMed

    Young, Kaylie L; Scott, Alexander W; Hao, Liangliang; Mirkin, Sarah E; Liu, Guoliang; Mirkin, Chad A

    2012-07-11

    Cellular transfection of nucleic acids is necessary for regulating gene expression through antisense or RNAi pathways. The development of spherical nucleic acids (SNAs, originally gold nanoparticles functionalized with synthetic oligonucleotides) has resulted in a powerful set of constructs that are able to efficiently transfect cells and regulate gene expression without the use of auxiliary cationic cocarriers. The gold core in such structures is primarily used as a template to arrange the nucleic acids into a densely packed and highly oriented form. In this work, we have developed methodology for coating the gold particle with a shell of silica, modifying the silica with a layer of oligonucleotides, and subsequently oxidatively dissolving the gold core with I(2). The resulting hollow silica-based SNAs exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold-core SNA counterparts. Importantly, they exhibit no cytotoxicity and have been used to effectively silence the eGFP gene in mouse endothelial cells through an antisense approach. PMID:22725653

  5. Membrane simulations mimicking acidic pH reveal increased thickness and negative curvature in a bilayer consisting of lysophosphatidylcholines and free fatty acids.

    PubMed

    Lähdesmäki, Katariina; Ollila, O H Samuli; Koivuniemi, Artturi; Kovanen, Petri T; Hyvönen, Marja T

    2010-05-01

    Phospholipids are key components of biological membranes and their lipolysis with phospholipase A(2) (PLA(2)) enzymes occurs in different cellular pH environments. Since no studies are available on the effect of pH on PLA(2)-modified phospholipid membranes, we performed 50-ns atomistic molecular dynamics simulations at three different pH conditions (pH 9.0, 7.5, and 5.5) using a fully PLA(2)-hydrolyzed phosphatidylcholine (PC) bilayer which consists solely of lysophosphatidylcholine and free fatty acid molecules. We found that a decrease in pH results in lateral squeezing of the membrane, i.e. in decreased surface area per headgroup. Thus, at the decreased pH, the lipid hydrocarbon chains had larger S(CD) order parameter values, and also enhanced membrane thickness, as seen in the electron density profiles across the membrane. From the lateral pressure profiles, we found that the values of spontaneous curvature of the two opposing monolayers became negative when the pH was decreased. At low pH, protonation of the free fatty acid headgroups reduces their mutual repulsion and accounts for the pH dependence of all the above-mentioned properties. The altered structural characteristics may significantly affect the overall surface properties of biomembranes in cellular vesicles, lipid droplets, and plasma lipoproteins, play an important role in membrane fission and fusion, and modify interactions between membrane lipids and the proteins embedded within them. PMID:20132791

  6. Splice cassette II of Na+,HCO3(-) cotransporter NBCn1 (slc4a7) interacts with calcineurin A: implications for transporter activity and intracellular pH control during rat artery contractions.

    PubMed

    Danielsen, Andreas A; Parker, Mark D; Lee, Soojung; Boron, Walter F; Aalkjaer, Christian; Boedtkjer, Ebbe

    2013-03-22

    Activation of Na(+),HCO3(-) cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH(i)) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na(+),HCO3(-) cotransporter NBCn1 (slc4a7) and the Ca(2+)/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pHi control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na(+),HCO3(-) cotransport activity was investigated in VSMCs of mesenteric arteries after an NH4(+) prepulse. During depolarization with 50 mM extracellular K(+) to raise intracellular [Ca(2+)], Na(+),HCO3(-) cotransport activity was inhibited 20-30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na(+),HCO3(-) cotransport activity in VSMCs when cytosolic [Ca(2+)] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na(+)/H(+) exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca(2+) activates Na(+),HCO3(-) cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification. PMID:23382378

  7. A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid.

    PubMed

    Ludwig-Müller, Jutta; Jülke, Sabine; Geiß, Kathleen; Richter, Franziska; Mithöfer, Axel; Šola, Ivana; Rusak, Gordana; Keenan, Sandi; Bulman, Simon

    2015-05-01

    The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae-infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids. PMID:25135243

  8. Intracellular Nucleic Acid Delivery by the Supercharged Dengue Virus Capsid Protein

    PubMed Central

    Freire, João Miguel; Veiga, Ana Salomé; Conceição, Thaís M.; Kowalczyk, Wioleta; Mohana-Borges, Ronaldo; Andreu, David; Santos, Nuno C.; Da Poian, Andrea T.; Castanho, Miguel A. R. B.

    2013-01-01

    Supercharged proteins are a recently identified class of proteins that have the ability to efficiently deliver functional macromolecules into mammalian cells. They were first developed as bioengineering products, but were later found in the human proteome. In this work, we show that this class of proteins with unusually high net positive charge is frequently found among viral structural proteins, more specifically among capsid proteins. In particular, the capsid proteins of viruses from the Flaviviridae family have all a very high net charge to molecular weight ratio (> +1.07/kDa), thus qualifying as supercharged proteins. This ubiquity raises the hypothesis that supercharged viral capsid proteins may have biological roles that arise from an intrinsic ability to penetrate cells. Dengue virus capsid protein was selected for a detailed experimental analysis. We showed that this protein is able to deliver functional nucleic acids into mammalian cells. The same result was obtained with two isolated domains of this protein, one of them being able to translocate lipid bilayers independently of endocytic routes. Nucleic acids such as siRNA and plasmids were delivered fully functional into cells. The results raise the possibility that the ability to penetrate cells is part of the native biological functions of some viral capsid proteins. PMID:24339931

  9. Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery.

    PubMed

    Yang, Yanfang; Xia, Xuejun; Dong, Wujun; Wang, Hongliang; Li, Lin; Ma, Panpan; Sheng, Wei; Xu, Xueqing; Liu, Yuling

    2016-05-01

    An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular environment and redox effect of glutathione in the cytosol, a new siRNA transporting system combining triple effects of folate targeting, acid sensitive polymer micelles, and bio-reducible disulfide bond linked siRNA-cell penetrating peptides (CPPs) conjugate is developed to suppress c-myc gene expression of breast cancer (MCF-7 cells) both in vitro and in vivo. Subsequent research demonstrates that the vesicle has particle size of about 100 nm and siRNA entrapment efficiency of approximately 80%. In vitro studies verified over 90% of encapsulated siRNA-CPPs can be released and the vesicle shows higher cellular uptake in response to the tumorous zone. Determination of gene expression at both mRNA and protein levels indicates the constructed vesicle exhibited enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo. PMID:26822264

  10. Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

    PubMed Central

    Citores, Lucía; Wesche, Jørgen; Kolpakova, Elona; Olsnes, Sjur

    1999-01-01

    Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment. PMID:10564275

  11. Reactive solute transport in an acidic stream: Experimental pH increase and simulation of controls on pH, aluminum, and iron

    USGS Publications Warehouse

    Broshears, R.E.; Runkel, R.L.; Kimball, B.A.; McKnight, Diane M.; Bencala, K.E.

    1996-01-01

    Solute transport simulations quantitatively constrained hydrologic and geochemical hypotheses about field observations of a pH modification in an acid mine drainage stream. Carbonate chemistry, the formation of solid phases, and buffering interactions with the stream bed were important factors in explaining the behavior of pH, aluminum, and iron. The precipitation of microcrystalline gibbsite accounted for the behavior of aluminum; precipitation of Fe(OH)3 explained the general pattern of iron solubility. The dynamic experiment revealed limitations on assumptions that reactions were controlled only by equilibrium chemistry. Temporal variation in relative rates of photoreduction and oxidation influenced iron behavior. Kinetic limitations on ferrous iron oxidation and hydrous oxide precipitation and the effects of these limitations on field filtration were evident. Kinetic restraints also characterized interaction between the water column and the stream bed, including sorption and desorption of protons from iron oxides at the sediment-water interface and post-injection dissolution of the precipitated aluminum solid phase.

  12. Rapid 3D Patterning of Poly(acrylic acid) Ionic Hydrogel for Miniature pH Sensors.

    PubMed

    Yin, Ming-Jie; Yao, Mian; Gao, Shaorui; Zhang, A Ping; Tam, Hwa-Yaw; Wai, Ping-Kong A

    2016-02-17

    Poly(acrylic acid) (PAA), as a highly ionic conductive hydrogel, can reversibly swell/deswell according to the surrounding pH conditions. An optical maskless -stereolithography technology is presented to rapidly 3D pattern PAA for device fabrication. A highly sensitive miniature pH sensor is demonstrated by in situ printing of periodic PAA micropads on a tapered optical microfiber. PMID:26643765

  13. Acid-base pH curves in vitro with mixtures of pure cultures of human oral microorganisms.

    PubMed

    Wijeyeweera, R L; Kleinberg, I

    1989-01-01

    Pure cultures of microorganisms commonly found in supragingival plaque were incubated alone and in combinations to determine the bacterial contribution to the pH-fall-pH-rise that is the central characteristic of the Stephan-curve pH change seen in plaque in vivo after brief exposure to a sugar solution. To avoid the complicating conditions of saliva flow and plaque diffusion, experiments were done with bacterial suspensions in incubations in vitro. In an initial experimental series where each microorganism was incubated only with glucose, all but a few produced the initial pH fall. Some also showed a subsequent small, sharp rise in the pH which then quickly levelled off; this was due to metabolism of endogenous substrate accumulated by most microorganisms during their growth in culture. When arginolytic and non-arginolytic bacteria were each then incubated with both glucose and arginine present (the glucose substrate to stimulate a pH fall and the arginine to stimulate a pH rise), the non-arginolytic gave a progressively more acidic pH response with progressive increase in the cell concentration, whereas the arginolytic bacteria produced a much smaller and variable pH decrease with similar cell concentration increase. Mixing pure cultures of either arginolytic or non-arginolytic bacteria gave acid-base pH responses similar to those of their respective pure cultures, whereas mixing arginolytic with non-arginolytic bacteria resulted in an approximate averaging of their different curves. The organisms present in highest proportion in a mixture had the greatest effects. The outcome of mixing the most numerous streptococcal and actinomyces species found normally in supragingival plaque indicated that the well-established difference in the acidity level of the Stephan pH response of caries-active and caries-inactive plaques could be due to differences in the proportions of their arginolytic and non-arginolytic members. PMID:2675801

  14. Influence of amino acids, buffers, and ph on the γ-irradiation-induced degradation of alginates.

    PubMed

    Ulset, Ann-Sissel T; Mori, Hideki; Dalheim, Marianne Ø; Hara, Masayuki; Christensen, Bjørn E

    2014-12-01

    Alginate-based biomaterials and medical devices are commonly subjected to γ-irradiation as a means of sterilization, either in the dry state or the gel (hydrated) state. In this process the alginate chains degrade randomly in a dose-dependent manner, altering alginates' material properties. The addition of free radical scavenging amino acids such as histidine and phenylalanine protects the alginate significantly against degradation, as shown by monitoring changes in the molecular weight distributions using SEC-MALLS and determining the pseudo first order rate constants of degradation. Tris buffer (0.5 M), but not acetate, citrate, or phosphate buffers had a similar effect on the degradation rate. Changes in pH itself had only marginal effects on the rate of alginate degradation and on the protective effect of amino acids. Contrary to previous reports, the chemical composition (M/G profile) of the alginates, including homopolymeric mannuronan, was unaltered following irradiation up to 10 kGy. PMID:25412478

  15. Lactic acid fermentation from food waste with indigenous microbiota: Effects of pH, temperature and high OLR.

    PubMed

    Tang, Jialing; Wang, Xiaochang; Hu, Yisong; Zhang, Yongmei; Li, Yuyou

    2016-06-01

    The effects of pH, temperature and high organic loading rate (OLR) on lactic acid production from food waste without extra inoculum addition were investigated in this study. Using batch experiments, the results showed that although the hydrolysis rate increased with pH adjustment, the lactic acid concentration and productivity were highest at pH 6. High temperatures were suitable for solubilization but seriously restricted the acidification processes. The highest lactic acid yield (0.46g/g-TS) and productivity (278.1mg/Lh) were obtained at 37°C and pH 6. In addition, the lactic acid concentration gradually increased with the increase in OLR, and the semi-continuous reactor could be stably operated at an OLR of 18g-TS/Ld. However, system instability, low lactic acid yield and a decrease in VS removal were noticed at high OLRs (22g-TS/Ld). The concentrations of volatile fatty acids (VFAs) in the fermentation mixture were relatively low but slightly increased with OLR, and acetate was the predominant VFA component. Using high-throughput pyrosequencing, Lactobacillus from the raw food waste was found to selectively accumulate and become dominant in the semi-continuous reactor. PMID:27040090

  16. First-principles calculation of thermodynamic stability of acids and bases under pH environment: A microscopic pH theory

    NASA Astrophysics Data System (ADS)

    Kim, Yong-Hyun; Kim, Kwiseon; Zhang, S. B.

    2012-04-01

    Despite being one of the most important thermodynamic variables, pH has yet to be incorporated into first-principles thermodynamics to calculate stability of acidic and basic solutes in aqueous solutions. By treating the solutes as defects in homogeneous liquids, we formulate a first-principles approach to calculate their formation energies under proton chemical potential, or pH, based on explicit molecular dynamics. The method draws analogy to first-principle calculations of defect formation energies under electron chemical potential, or Fermi energy, in semiconductors. From this, we propose a simple pictorial representation of the general theory of acid-base chemistry. By performing first-principles molecular dynamics of liquid water models with solutes, we apply the formulation to calculate formation energies of various neutral and charged solutes such as H+, OH-, NH3, NH4+, HCOOH, and HCOO- in water. The deduced auto-dissociation constant of water and the difference in the pKa values of NH3 and HCOOH show good agreement with known experimental values. Our first-principles approach can be further extended and applied to other bio- and electro-chemical molecules such as amino acids and redox reaction couples that could exist in aqueous environments to understand their thermodynamic stability.

  17. First-Principles Calculation of Thermodynamic Stability of Acids and Bases under pH Environment: A Microscopic pH Theory

    SciTech Connect

    Kim, Y. H.; Kim, K.; Zhang, S. B.

    2012-04-07

    Despite being one of the most important thermodynamic variables, pH has yet to be incorporated into first-principles thermodynamics to calculate stability of acidic and basic solutes in aqueous solutions. By treating the solutes as defects in homogeneous liquids, we formulate a first-principles approach to calculate their formation energies under proton chemical potential, or pH, based on explicit molecular dynamics. The method draws analogy to first-principle calculations of defect formation energies under electron chemical potential, or Fermi energy, in semiconductors. From this, we propose a simple pictorial representation of the general theory of acid-base chemistry. By performing first-principles molecular dynamics of liquid water models with solutes, we apply the formulation to calculate formation energies of various neutral and charged solutes such as H{sup +}, OH{sup -}, NH{sub 3}, NH{sub 4}{sup +}, HCOOH, and HCOO{sup -} in water. The deduced auto-dissociation constant of water and the difference in the pKa values of NH{sub 3} and HCOOH show good agreement with known experimental values. Our first-principles approach can be further extended and applied to other bio- and electro-chemical molecules such as amino acids and redox reaction couples that could exist in aqueous environments to understand their thermodynamic stability.

  18. Temperature and pH responsiveness of poly-(DMAA-co-unsaturated carboxylic acid) hydrogels synthesized by UV-irradiation

    NASA Astrophysics Data System (ADS)

    Kakinoki, Sachiro; Kaetsu, Isao; Nakayama, Masashi; Sutani, Kouichi; Uchida, Kumao; Yukutake, Kouji

    2003-07-01

    Stimuli-responsive polyampholyte hydrogels were synthesized by the copolymerization of dimethylaminoethyl methacrylate (DMAA) and acrylic acid (AAc) or itaconic acid (IAc) by UV-irradiation. Temperature and pH responsiveness of these hydrogels were studied. The temperature responsiveness of poly-(DMAA-co-AAc, IAc) hydrogels shown in change of water content became dull compared to that of DMAA homo-polymer hydrogel. The water content of the poly-(DMAA-co-AAc, IAc) hydrogels showed a minimum at pH 8, and increased in more acidic and alkaline regions. This fact can be attributed to the coexistence of anions and cations in the poly-(DMAA-co-AAc, IAc) hydrogels. The poly-(DMAA-co-AAc, IAc) hydrogels were polyampholyte having both temperature responsiveness and pH responsiveness.

  19. [Characteristics of soil pH and exchangeable acidity in red soil profile under different vegetation types].

    PubMed

    Ji, Gang; Xu, Ming-gang; Wen, Shi-lin; Wang, Bo-ren; Zhang, Lu; Liu, Li-sheng

    2015-09-01

    The characteristics of soil pH and exchangeable acidity in soil profile under different vegetation types were studied in hilly red soil regions of southern Hunan Province, China. The soil samples from red soil profiles within 0-100 cm depth at fertilized plots and unfertilized plots were collected and analyzed to understand the profile distribution of soil pH and exchangeable acidity. The results showed that, pH in 0-60 cm soil from the fertilized plots decreased as the following sequence: citrus orchard > Arachis hypogaea field > tea garden. As for exchangeable acidity content, the sequence was A. hypogaea field ≤ citrus orchard < tea garden. After tea tree and A. hypogaea were planted for long time, acidification occurred in surface soil (0-40 cm), compared with the deep soil (60-100 cm), and soil pH decreased by 0.55 and 0.17 respectively, but such changes did not occur in citrus orchard. Soil pH in 0-40 cm soil from the natural recovery vegetation unfertilized plots decreased as the following sequence: Imperata cylindrica land > Castanea mollissima garden > Pinus elliottii forest ≥ Loropetalum chinensis forest. As for exchangeable acidity content, the sequence was L cylindrica land < C. mollissima garden < L. chinensis forest ≤ P. elliottii forest. Soil pH in surface soil (0-20 cm) from natural forest plots, secondary forest and Camellia oleifera forest were significantly lower than that from P. massoniana forest, decreased by 0.34 and 0.20 respectively. For exchangeable acidity content in 0-20 cm soil from natural forest plot, P. massoniana forest and secondary forest were significantly lower than C. oleifera forest. Compared with bare land, surface soil acidification in unfertilized plots except I. cylindrica land had been accelerated, and the natural secondary forest was the most serious among them, with surface soil pH decreasing by 0.52. However, the pH increased in deep soils from unfertilized plots except natural secondary forest, and I. cylindrica

  20. Development of On-Line Spectroscopic pH Monitoring for Nuclear Fuel Reprocessing Plants: Weak Acid Schemes

    SciTech Connect

    Casella, Amanda J.; Hylden, Laura R.; Campbell, Emily L.; Levitskaia, Tatiana G.; Peterson, James M.; Smith, Frances N.; Bryan, Samuel A.

    2015-05-19

    Knowledge of real-time solution properties and composition is a necessity for any spent nuclear fuel reprocessing method. Metal-ligand speciation in aqueous solutions derived from the dissolved commercial spent fuel is highly dependent upon the acid concentration/pH, which influences extraction efficiency and the resulting speciation in the organic phase. Spectroscopic process monitoring capabilities, incorporated in a counter current centrifugal contactor bank, provide a pathway for on-line real-time measurement of solution pH. The spectroscopic techniques are process-friendly and can be easily configured for on-line applications, while classic potentiometric pH measurements require frequent calibration/maintenance and have poor long-term stability in aggressive chemical and radiation environments. Our research is focused on developing a general method for on-line determination of pH of aqueous solutions through chemometric analysis of Raman spectra. Interpretive quantitative models have been developed and validated under the range of chemical composition and pH using a lactic acid/lactate buffer system. The developed model was applied to spectra obtained on-line during solvent extractions performed in a centrifugal contactor bank. The model predicted the pH within 11% for pH > 2, thus demonstrating that this technique could provide the capability of monitoring pH on-line in applications such as nuclear fuel reprocessing.

  1. Combined stress effect of pH and temperature narrows the niche width of flagellates in acid mining lakes

    PubMed Central

    Moser, Michael; Weisse, Thomas

    2011-01-01

    Strains of the green alga Chlamydomonas acidophila and two chrysomonads, Ochromonas spp., isolated from each of two similar acid mining lakes (AMLs) with extremely low pH (∼2.6) were investigated to consider a possible synergistic stress effect of low pH and unfavourable temperature. We measured flagellate growth rates over a combination of four pH (2.5, 3.5, 5.0 and 7.0) and three temperatures (10, 17.5 and 25°C) in the laboratory. Our hypothesis was that, under highly acidic conditions (pH <3), an obligate acidophil species (C. acidophila) would be less sensitive to the combined stress of pH and temperature than acidotolerant species (Ochromonas spp.). We expected that the difference of the fundamental vs. realized pH niche would be greater in the latter. Another chrysomonad, Poterioochromonas malhamensis strain DS, served as a reference for a closely related neutrophil species. Surprisingly, C. acidophila did not survive temperatures >27°C. The lowest temperature tested reduced growth rates of all three chrysomonad strains significantly. Since all chrysomonads were tolerant to high temperature, growth rate of one Ochromonas spp. strain was measured exemplarily at 35°C. Only at this high temperature was the realized pH niche significantly narrowed. We also recorded significant intraspecific differences within the C. acidophila strains from the two AML, illustrating that the niche width of a species is broader than that of individual clones. PMID:21655470

  2. Selection method of pH conditions to establish Pseudomonas taetrolens physiological states and lactobionic acid production.

    PubMed

    Alonso, Saúl; Rendueles, Manuel; Díaz, Mario

    2013-05-01

    Microbial physiological responses resulting from inappropriate bioprocessing conditions may have a marked impact on process performance within any fermentation system. The influence of different pH-control strategies on physiological status, microbial growth and lactobionic acid production from whey by Pseudomonas taetrolens during bioreactor cultivations has been investigated for the first time in this work. Both cellular behaviour and bioconversion efficiency from P. taetrolens were found to be negatively influenced by pH-control modes carried out at values lower than 6.0 and higher than 7.0. Production schemes were also influenced by the operational pH employed, with asynchronous production from damaged and metabolically active subpopulations at pH values lower than 6.0. Moreover, P. taetrolens showed reduced cellular proliferation and a subsequent delay in the onset of the production phase under acidic conditions (pH < 6.0). Unlike cultivations performed at 6.5, both pH-shift and pH-stat cultivation strategies performed at pH values lower than 6.0 resulted in decreased lactobionic acid production. Whereas the cellular response showed a stress-induced physiological response under acidic conditions, healthy functional cells were predominant at medium operational pH values (6.5-7.0). P. taetrolens thus displayed a robust physiological status at initial pH value of 6.5, resulting in an enhanced bioconversion yield and lactobionic acid productivity (7- and 4-fold higher compared to those attained at initial pH values of 4.5 and 5.0, respectively). These results have shown that pH-control modes strongly affected both the physiological response of cells and the biological performance of P. taetrolens, providing key information for bio-production of lactobionic acid on an industrial scale. PMID:23254761

  3. Hydrothermal carbonization (HTC) of wheat straw: influence of feedwater pH prepared by acetic acid and potassium hydroxide.

    PubMed

    Reza, M Toufiq; Rottler, Erwin; Herklotz, Laureen; Wirth, Benjamin

    2015-04-01

    In this study, influence of feedwater pH (2-12) was studied for hydrothermal carbonization (HTC) of wheat straw at 200 and 260°C. Acetic acid and KOH were used as acidic and basic medium, respectively. Hydrochars were characterized by elemental and fiber analyses, SEM, surface area, pore volume and size, and ATR-FTIR, while HTC process liquids were analyzed by HPLC and GC. Both hydrochar and HTC process liquid qualities vary with feedwater pH. At acidic pH, cellulose and elemental carbon increase in hydrochar, while hemicellulose and pseudo-lignin decrease. Hydrochars produced at pH 2 feedwater has 2.7 times larger surface area than that produced at pH 12. It also has the largest pore volume (1.1 × 10(-1) ml g(-1)) and pore size (20.2 nm). Organic acids were increasing, while sugars were decreasing in case of basic feedwater, however, phenolic compounds were present only at 260°C and their concentrations were increasing in basic feedwater. PMID:25710573

  4. Metal reduction at low pH by a Desulfosporosinus species: implications for the biological treatment of acidic mine drainage

    SciTech Connect

    Senko, J.M.; Zhang, G.X.; McDonough, J.T.; Bruns, M.A.; Burgos, W.D.

    2009-07-01

    We isolated an acid-tolerant sulfate-reducing bacterium, GBSRB4.2, from coal mine-derived acidic mine drainage (AMD)-derived sediments. Sequence analysis of partial 16S rRNA gene of GBSRB4.2 revealed that it was affiliated with the genus Desulfosporosinus. GBSRB4.2 reduced sulfate, Fe(III) (hydr)oxide, Mn(IV) oxide, and U(VI) in acidic solutions (pH 4.2). Sulfate, Fe(III), and Mn(IV) but not U(VI) bioreduction led to an increase in the pH of acidic solutions and concurrent hydrolysis and precipitation of dissolved Al{sup 3+}. Reduction of Fe(III), Mn(IV), and U(VI) in sulfate-free solutions revealed that these metals are enzymatically reduced by GBSRB4.2. GBSRB4.2 reduced U(VI) in groundwater from a radionuclide-contaminated aquifer more rapidly at pH 4.4 than at pH 7.1, possibly due to the formation of poorly bioreducible Ca-U(VI)-CO{sub 3} complexes in the pH 7.1 groundwater.

  5. Acidic Nanoparticles Are Trafficked to Lysosomes and Restore an Acidic Lysosomal pH and Degradative Function to Compromised ARPE-19 Cells

    PubMed Central

    Baltazar, Gabriel C.; Guha, Sonia; Lu, Wennan; Lim, Jason; Boesze-Battaglia, Kathleen; Laties, Alan M.; Tyagi, Puneet; Kompella, Uday B.; Mitchell, Claire H.

    2012-01-01

    Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide) (PLGA) 502 H, PLGA 503 H and poly (DL-lactide) (PLA) colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity. PMID:23272048

  6. Proton-dependent zinc release from intracellular ligands.

    PubMed

    Kiedrowski, Lech

    2014-07-01

    In cultured cortical and hippocampal neurons when intracellular pH drops from 6.6 to 6.1, yet unclear intracellular stores release micromolar amounts of Zn(2+) into the cytosol. Mitochondria, acidic organelles, and/or intracellular ligands could release this Zn(2+) . Although exposure to the protonophore FCCP precludes reloading of the mitochondria and acidic organelles with Zn(2+) , FCCP failed to compromise the ability of the intracellular stores to repeatedly release Zn(2+) . Therefore, Zn(2+) -releasing stores were not mitochondria or acidic organelles but rather intracellular Zn(2+) ligands. To test which ligands might be involved, the rate of acid-induced Zn(2+) release from complexes with cysteine, glutathione, histidine, aspartate, glutamate, glycine, and carnosine was investigated; [Zn(2+) ] was monitored in vitro using the ratiometric Zn(2+) -sensitive fluorescent probe FuraZin-1. Carnosine failed to chelate Zn(2+) but did chelate Cu(2+) ; the remaining ligands chelated Zn(2+) and upon acidification were releasing it into the medium. However, when pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes rapidly accelerated the rate of Zn(2+) release. The zinc-cysteine complexes also released Zn(2+) when a histidine-modifying agent, diethylpyrocarbonate, was applied at pH 7.2. Since the cytosolic zinc-cysteine complexes can contain micromolar amounts of Zn(2+) , these complexes may represent the stores responsible for an acid-induced intracellular Zn(2+) release. This study aimed at identifying intracellular stores which release Zn(2+) when pHi drops from 6.6 to 6.1. It was found that these stores are not mitochondria or acidic organelles, but rather intracellular Zn(2+) ligands. When the pH was decreasing from 6.6 to 6.1, only zinc-cysteine complexes showed a rapid acceleration in the rate of Zn(2+) release. Therefore, the stores responsible for an acid-induced intracellular Zn(2+) release in neurons may be the cytosolic zinc-cysteine complexes

  7. The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

    NASA Technical Reports Server (NTRS)

    Cleland, R. E.; Buckley, G.; Nowbar, S.; Lew, N. M.; Stinemetz, C.; Evans, M. L.; Rayle, D. L.

    1991-01-01

    The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxin-treated tissues (4.5.-5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5-6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.

  8. Intracellular Uptake and Trafficking of Difluoroboron Dibenzoylmethane-Poly(lactic acid) Nanoparticles in HeLa Cells

    PubMed Central

    Contreras, Janette; Xie, Jiansong; Chen, Yin Jie; Pei, Hua; Zhang, Guoqing; Fraser, Cassandra L.; Hamm-Alvarez, Sarah F.

    2010-01-01

    In this study, nanoparticles based on difluoroboron dibenzoylmethane-poly(lactic acid) (BF2dbmPLA) are prepared. Polylactic acid or polylactide is a commonly used degradable polymer, while the boron dye possesses a large extinction coefficient, high emission quantum yield, 2-photon absorption, and sensitivity to the surrounding environment. BF2dbmPLA exhibits molecular weight-dependent emission properties, and can be formulated as stable nanoparticles, suggesting that its unique optical properties may be useful in multiple contexts for probing intracellular environments. Here we show that BF2dbmPLA nanoparticles are internalized into cultured HeLa cells by endocytosis, and that within the cellular milieu they retain their fluorescence properties. BF2dbmPLA nanoparticles are photostable, resisting laser-induced photobleaching under conditions that destroy the fluorescence of a common photostable probe, LysoTracker™ blue. Their endocytosis is also lipid raft-dependent, as evidenced by their significant co-localization with cholera toxin B subunit in membrane compartments after uptake, and their sensitivity of uptake to methyl-β-cyclodextrin. Additionally, BF2dbmPLA nanoparticle endocytosis utilizes microtubules and actin filaments. Internalized BF2dbmPLA nanoparticles do not accumulate in acidic late endosomes and lysosomes, but within a perinuclear non-lysosomal compartment. These findings demonstrate the feasibility of using novel BF2dbmPLA nanoparticles exhibiting diverse emission properties for in situ, live cell imaging, and suggest that their endogenous uptake occurs through a lipid-raft dependent endocytosis mechanism. PMID:20420413

  9. Preparation, Characterization and Intracellular Imaging of 2,4-Dichlorophenoxyacetic Acid Conjugated Gold Nanorods.

    PubMed

    Jia, Jin-Liang; Jin, Xiao-Yong; Liu, Qing-Le; Liang, Wen-Long; Lin, Miao-Shan; Xu, Han-Hong

    2016-05-01

    Visualizing the biodistribution of pesticides inside living cells is great importance for enhancing targeting of pesticides. Here we reported for the first time that gold nanorods (Au NRs) with size of 39.4 nm x 11.3 nm could be used as a fluorescent tracer to examine the distribution of a typical herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), in tobacco bright yellow 2 (BY-2) cells. The nanostructures of hybrid materials were analyzed by using Raman spectra and X-ray photoelectron spectroscopy (XPS), including spectra assignments and electronic property. These data revealed 2,4-D has successfully conjugated MP-Au NRs according to Raman and XPS. The biodistribution of the conjugates inside BY-2 cells was directly examined at 12 and 24 h by the two-photon microscopy. The intensity of two-photon luminescence (TPL) inside cells demonstrated that the conjugates could be localized and excluded by BY-2 cells. Thus, this labeling approach opens up new avenues to the facile and efficient labeling of pesticides. PMID:27483849

  10. Simultaneous determination of intracellular UDP-sugars in hyaluronic acid-producing Streptococcus zooepidemicus.

    PubMed

    Franke, Lukáš; Čožíková, Dagmar; Smirnou, Dzianis; Hermannová, Martina; Hanová, Tereza; Růžičková, Andrea; Velebný, Vladimír

    2015-08-01

    Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells. PMID:26114654

  11. Growth and Metabolism of Lactic Acid Bacteria during and after Malolactic Fermentation of Wines at Different pH

    PubMed Central

    Davis, C. R.; Wibowo, D. J.; Lee, T. H.; Fleet, G. H.

    1986-01-01

    Commercially produced red wines were adjusted to pH 3.0, 3.2, 3.5, 3.7, or 4.0 and examined during and after malolactic fermentation for growth of lactic acid bacteria and changes in the concentrations of carbohydrates, organic acids, amino acids, and acetaldehyde. With one exception, Leuconostoc oenos conducted the malolactic fermentation in all wines and was the only species to occur in wines at pH below 3.5. Malolactic fermentation by L. oenos was accompanied by degradation of malic, citric, and fumaric acids and production of lactic and acetic acids. The concentrations of arginine, histidine, and acetaldehyde also decreased at this stage, but the behavior of hexose and pentose sugars was complicated by other factors. Pediococcus parvulus conducted the malolactic fermentation in one wine containing 72 mg of total sulfur dioxide per liter. Fumaric and citric acids were not degraded during this malolactic fermentation, but hexose sugars were metabolized. P. parvulus and species of Lactobacillus grew after malolactic fermentation in wines with pH adjusted above 3.5. This growth was accompanied by the utilization of wine sugars and production of lactic and acetic acids. PMID:16347015

  12. Denitrification in Streams Impacted by Acid Mine Drainage: Effects of Iron, pH, and Potential Electron Donors

    NASA Astrophysics Data System (ADS)

    Baeseman, J. L.; Smith, R. L.; Silverstein, J.

    2003-12-01

    Acid mine drainage (AMD) contaminates between 8,000 and 16,000 km of streams on U.S. Forest Service land in the Western United States and more than 7,000 km of stream in the Eastern U.S. Relatively little is known about nitrogen cycling in these acidic, heavy metal laden streams, however, denitrification can be inhibited under low pH conditions. The objective of this research was to examine AMD sediments for bacteria capable of denitrification. The process of denitrification is known to increase pH, which may be particularly important in acidic environments. Denitrification potential was assessed in AMD sediments from several Colorado AMD impacted streams ranging from pH 2.6 to 4.91, using microcosm incubations with fresh sediments. Added nitrate was immediately reduced to nitrogen gas without any lag period, indicating that denitrification was actively occurring in these environments. Rates varied from 0.33 to 2.52 umoles NO3-N/ g-sediment/ day depending on the site. The pH of the microcosms increased between 0.23 to 1.49 pH units in 5 days, depending on the site. Additional microcosm studies were conducted to examine the effects of iron concentrations (Fe2+ and Fe3+), initial pH conditions, and several potential electron donors. Addition of iron above ambient concentrations seemed to have little effect on denitrification rates, whereas rates increased with increasing initial pH. The addition of carbon and hydrogen stimulated denitrification rates, which in turn increased the rise in pH. These results suggest that not only is denitrification possible in AMD streams, it may also be a useful remediation option, if suitable methods can be found to stimulate activity.

  13. Anti-biofilm potential of phenolic acids: the influence of environmental pH and intrinsic physico-chemical properties.

    PubMed

    Silva, Sara; Costa, Eduardo M; Horta, Bruno; Calhau, Conceição; Morais, Rui M; Pintado, M Manuela

    2016-09-13

    Phenolic acids are a particular group of small phenolic compounds which have exhibited some anti-biofilm activity, although the link between their activity and their intrinsic pH is not clear. Therefore, the present work examined the anti-biofilm activity (inhibition of biomass and metabolic activity) of phenolic acids in relation to the environmental pH, as well as other physico-chemical properties. The results indicate that, while Escherichia coli was not inhibited by the phenolic acids, both methicillin resistant Staphylococcus aureus and methicillin resistant Staphylococcus epidermidis were susceptible to the action of all phenolic acids, with the pH playing a relevant role in the activity: a neutral pH favored MRSE inhibition, while acidic conditions favored MRSA inhibition. Some links between molecular polarity and size were associated only with their potential as metabolic inhibitors, with the overall interactions hinting at a membrane-based mechanism for MRSA and a cytoplasmic effect for MRSE. PMID:27434592

  14. Effect of acid rain pH on leaching behavior of cement stabilized lead-contaminated soil.

    PubMed

    Du, Yan-Jun; Wei, Ming-Li; Reddy, Krishna R; Liu, Zhao-Peng; Jin, Fei

    2014-04-30

    Cement stabilization is a practical approach to remediate soils contaminated with high levels of lead. However, the potential for leaching of lead out of these stabilized soils under variable acid rain pH conditions is a major environmental concern. This study investigates the effects of acid rain on the leaching characteristics of cement stabilized lead contaminated soil under different pH conditions. Clean kaolin clay and the same soil spiked with 2% lead contamination are stabilized with cement contents of 12 and 18% and then cured for 28 days. The soil samples are then subjected to a series of accelerated leaching tests (or semi-dynamic leaching tests) using a simulated acid rain leachant prepared at pH 2.0, 4.0 or 7.0. The results show that the strongly acidic leachant (pH ∼2.0) significantly altered the leaching behavior of lead as well as calcium present in the soil. However, the differences in the leaching behavior of the soil when the leachant was mildly acidic (pH ∼4.0) and neutral (pH ∼7.0) prove to be minor. In addition, it is observed that the lead contamination and cement content levels can have a considerable impact on the leaching behavior of the soils. Overall, the leachability of lead and calcium is attributed to the stability of the hydration products and their consequent influence on the soil buffering capacity and structure. PMID:24637445

  15. Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

    NASA Astrophysics Data System (ADS)

    Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

    1999-07-01

    Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

  16. ACCURACY OF ROSS PH COMBINATION ELECTRODES IN DILUTE SULPHURIC ACID STANDARDS

    EPA Science Inventory

    The mean observed pH of a 5.00 plus or minus 0.05 x 0.00001 M H2504 solution was 4.06 plus or minus 0.05 (2s) for 485 pH measurements by seven different operators, using nine Orion Ross Model 81-94b pH combination electrodes and four different pH meters over 8 weeks. Traditional ...

  17. Effects of acetic acid and arginine on pH elevation and growth of Bacillus licheniformis in an acidified cucumber juice medium.

    PubMed

    Yang, Zhenquan; Meng, Xia; Breidt, Frederick; Dean, Lisa L; Arritt, Fletcher M

    2015-04-01

    Bacillus licheniformis has been shown to cause pH elevation in tomato products having an initial pH below 4.6 and metabiotic effects that can lead to the growth of pathogenic bacteria. Because of this, the organism poses a potential risk to acidified vegetable products; however, little is known about the growth and metabolism of this organism in these products. To clarify the mechanisms of pH change and growth of B. licheniformis in vegetable broth under acidic conditions, a cucumber juice medium representative of a noninhibitory vegetable broth was used to monitor changes in pH, cell growth, and catabolism of sugars and amino acids. For initial pH values between pH 4.1 to 6.0, pH changes resulted from both fermentation of sugar (lowering pH) and ammonia production (raising pH). An initial pH elevation occurred, with starting pH values of pH 4.1 to 4.9 under both aerobic and anaerobic conditions, and was apparently mediated by the arginine deiminase reaction of B. licheniformis. This initial pH elevation was prevented if 5 mM or greater acetic acid was present in the brine at the same pH. In laboratory media, under favorable conditions for growth, data indicated that growth of the organism was inhibited at pH 4.6 with protonated acetic acid concentrations of 10 to 20 mM, corresponding to 25 to 50 mM total acetic acid; however, growth inhibition required greater than 300 mM citric acid (10-fold excess of the amount in processed tomato products) products under similar conditions. The data indicate that growth and pH increase by B. licheniformis may be inhibited by the acetic acid present in most commercial acidified vegetable products but not by the citric acid in many tomato products. PMID:25836398

  18. Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner.

    PubMed

    Kottyan, Leah C; Collier, Ann R; Cao, Khanh H; Niese, Kathryn A; Hedgebeth, Megan; Radu, Caius G; Witte, Owen N; Khurana Hershey, Gurjit K; Rothenberg, Marc E; Zimmermann, Nives

    2009-09-24

    The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5'-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclase-dependent manner. Furthermore, we identify G protein-coupled receptor 65 (GPR65) as the chief acid-sensing receptor expressed by eosinophils, as GPR65-deficient eosinophils were resistant to acid-induced eosinophil cAMP production and enhanced viability. Notably, GPR65(-/-) mice had attenuated airway eosinophilia and increased apoptosis in 2 distinct models of allergic airway disease. We conclude that eosinophil viability is increased in acidic microenvironments in a cAMP- and GPR65-dependent manner. PMID:19641187

  19. Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner

    PubMed Central

    Kottyan, Leah C.; Collier, Ann R.; Cao, Khanh H.; Niese, Kathryn A.; Hedgebeth, Megan; Radu, Caius G.; Witte, Owen N.; Khurana Hershey, Gurjit K.; Rothenberg, Marc E.

    2009-01-01

    The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5′-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclase–dependent manner. Furthermore, we identify G protein-coupled receptor 65 (GPR65) as the chief acid-sensing receptor expressed by eosinophils, as GPR65-deficient eosinophils were resistant to acid-induced eosinophil cAMP production and enhanced viability. Notably, GPR65−/− mice had attenuated airway eosinophilia and increased apoptosis in 2 distinct models of allergic airway disease. We conclude that eosinophil viability is increased in acidic microenvironments in a cAMP- and GPR65-dependent manner. PMID:19641187

  20. Gallic Acid as a Complexing Agent for Copper Chemical Mechanical Polishing Slurries at Neutral pH

    NASA Astrophysics Data System (ADS)

    Kim, Yung Jun; Kang, Min Cheol; Kwon, Oh Joong; Kim, Jae Jeong

    2011-05-01

    Gallic acid was investigated as a new complexing agent for copper (Cu) chemical mechanical polishing slurries at neutral pH. Addition of 0.03 M gallic acid and 1.12 M H2O2 at pH 7 resulted in a Cu removal rate of 560.73±17.49 nm/min, and the ratio of the Cu removal rate to the Cu dissolution rate was 14.8. Addition of gallic acid improved the slurry performance compared to glycine addition. X-ray photoelectron spectroscopy analysis and contact angle measurements showed that addition of gallic acid enhanced the Cu polishing behavior by suppressing the formation of surface Cu oxide.

  1. Chemical crosslinking of acrylic acid to form biocompatible pH sensitive hydrogel reinforced with cellulose nanocrystals (CNC)

    SciTech Connect

    Lim, Lim Sze; Ahmad, Ishak; Lazim, Mohd Azwani Shah Mat; Amin, Mohd. Cairul Iqbal Mohd

    2014-09-03

    The purpose of this study is to produce a novel pH and temperature sensitive hydrogel, composed of poly(acrylic acid) (PAA) and cellulose nanocrystal (CNC). CNC was extracted from kenaf fiber through a series of alkali and bleaching treatments followed by acid hydrolysis. The PAA was then subjected to chemical cross-linking using the cross-linking agent (N,N-methylenebisacrylamide) with CNC entrapped in PAA matrix. The mixture was casted onto petri dish to obtain disc shape hydrogel. The effects of reaction conditions such as the ratio of PAA and CNC on the swelling behavior of the hydrogel obtained towards pH and temperature were studied. The obtained hydrogel was further subjected to different tests such swelling test for swelling behaviour at different pH and temperature along with scanning electron microscopy (SEM) for morphology analysis. The hydrogel obtained showed excellent pH sensitivity and obtained maximum swelling at pH 7. Besides that, hydrogel obtained showed significant increase in swelling ratio when temperature of swelling medium was increased from 25°C to 37°C. SEM micrograph showed that the pore size of the hydrogel decreases with increase of CNC content proving that the hydrogel structure became more rigid with addition of CNC. The PAA/CNC hydrogel with such excellent sensitivity towards pH and temperature can be developed further as drug carrier.

  2. Chemical crosslinking of acrylic acid to form biocompatible pH sensitive hydrogel reinforced with cellulose nanocrystals (CNC)

    NASA Astrophysics Data System (ADS)

    Lim, Lim Sze; Ahmad, Ishak; Lazim, Mohd Azwani Shah Mat; Amin, Mohd. Cairul Iqbal Mohd

    2014-09-01

    The purpose of this study is to produce a novel pH and temperature sensitive hydrogel, composed of poly(acrylic acid) (PAA) and cellulose nanocrystal (CNC). CNC was extracted from kenaf fiber through a series of alkali and bleaching treatments followed by acid hydrolysis. The PAA was then subjected to chemical cross-linking using the cross-linking agent (N,N-methylenebisacrylamide) with CNC entrapped in PAA matrix. The mixture was casted onto petri dish to obtain disc shape hydrogel. The effects of reaction conditions such as the ratio of PAA and CNC on the swelling behavior of the hydrogel obtained towards pH and temperature were studied. The obtained hydrogel was further subjected to different tests such swelling test for swelling behaviour at different pH and temperature along with scanning electron microscopy (SEM) for morphology analysis. The hydrogel obtained showed excellent pH sensitivity and obtained maximum swelling at pH 7. Besides that, hydrogel obtained showed significant increase in swelling ratio when temperature of swelling medium was increased from 25°C to 37°C. SEM micrograph showed that the pore size of the hydrogel decreases with increase of CNC content proving that the hydrogel structure became more rigid with addition of CNC. The PAA/CNC hydrogel with such excellent sensitivity towards pH and temperature can be developed further as drug carrier.

  3. Factors influencing the acid-base (pH) balance in the Baltic Sea: a sensitivity analysis

    NASA Astrophysics Data System (ADS)

    Omstedt, Anders; Edman, Moa; Anderson, Leif G.; Laudon, Hjalmar

    2010-09-01

    Using calculations based on the marine carbon system and on modelling, the sensitivity of Baltic Sea surface pH was examined. Transient long-term calculations demonstrated that the marine carbon system adjusts to lateral boundary conditions within some decades, as does salinity. Climate changes in temperature or salinity will only marginally affect the acid-base (pH) balance. Wetter or dryer climate will also play a minor role in the pH balance. The direct effect on seawater pH of acid precipitation over the Baltic Sea surface was demonstrated to be small. Acidification due to river transport of dissolved organic carbon (DOC) into the marine system seems marginal although mineralization of terrestrial DOC may cause extra marine acidification, but the effect has yet to be quantified. Increased nutrient load may increase the amplitude in the pH seasonal cycle and increase the acidification during winter time. Fossil fuel burning is likely to have both a direct and indirect effect through increased CO2 levels, altering seawater pH as well as changing the river chemistry. This may severely threaten some species in the Baltic Sea, particularly in the Northern Baltic.

  4. Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes*

    PubMed Central

    Cao, Xu; Yang, Fan; Zheng, Jie; Wang, KeWei

    2012-01-01

    α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders. PMID:22679014

  5. Effect of low molecular weight organic acids on lowing pH in the sea water of the Jiaozhou Bay

    NASA Astrophysics Data System (ADS)

    Ding, H.; Zhou, Y.; Yang, G.; Lv, L.

    2013-12-01

    Recent study showed that average pH value in the seawater of the Jiaozhou Bay and its adjacent area of the Yellow Sea were about 7.9 and 8.0-8.2, respectively, indicating significant low pH value in the sea water of the bay. At the same period, existence of high concentrations of low molecular weight organic acids, including formate, acetate and lactate was detected. By theoretical calculation, field and laboratory simulate experiments, this study investigated the effect of these organic acids on pH value of the seawater in the Jiaozhou Bay. The results showed that average concentration of the total low molecular weight organic acids was 29.01 μmol/L; and average concentrations of formate, acetate and lactate were 4.06 μ mol/L, 18.31 μmol/L, and 6.64 μmol/L, respectively, in the surface seawater samples collected from 15 sampling stations in the Jiaozhou Bay in May, 2012. With similar total alkalinity (TA) and concentration of dissolved inorganic carbon (DIC) in the Jiaozhou Bay and the Yellow Sea, all the low molecular weight organic acids could decrease pH value in the seawater. Under field condition, co-effect of the three organic acids could decrease pH value in the sea water of the Jiaozhou Bay up to 0.185. We also collected samples of 6 stations of Narragansett Bay as comparison. The results supported that low molecular weight organic acids was critical on acidification of seawater in the Jiaozhou bay.

  6. Tyramine biosynthesis is transcriptionally induced at low pH and improves the fitness of Enterococcus faecalis in acidic environments.

    PubMed

    Perez, Marta; Calles-Enríquez, Marina; Nes, Ingolf; Martin, Maria Cruz; Fernandez, Maria; Ladero, Victor; Alvarez, Miguel A

    2015-04-01

    Enterococcus faecalis is a commensal bacterium of the human gut that requires the ability to pass through the stomach and therefore cope with low pH. E. faecalis has also been identified as one of the major tyramine producers in fermented food products, where they also encounter acidic environments. In the present work, we have constructed a non-tyramine-producing mutant to study the role of the tyramine biosynthetic pathway, which converts tyrosine to tyramine via amino acid decarboxylation. Wild-type strain showed higher survival in a system that mimics gastrointestinal stress, indicating that the tyramine biosynthetic pathway has a role in acid resistance. Transcriptional analyses of the E. faecalis V583 tyrosine decarboxylase cluster showed that an acidic pH, together with substrate availability, induces its expression and therefore the production of tyramine. The protective role of the tyramine pathway under acidic conditions appears to be exerted through the maintenance of the cytosolic pH. Tyramine production should be considered important in the adaptability of E. faecalis to acidic environments, such as fermented dairy foods, and to survive passage through the human gastrointestinal tract. PMID:25529314

  7. Acid-coated Textiles (pH 5.5-6.5)--a New Therapeutic Strategy for Atopic Eczema?

    PubMed

    Jaeger, Teresa; Rothmaier, Markus; Zander, Holger; Ring, Johannes; Gutermuth, Jan; Anliker, Mark D

    2015-07-01

    Increased transepidermal water loss (TEWL) and decreased skin capacitance are characteristic features of the disturbed epidermal barrier in atopic eczema (AE). The "acid mantle", which is a slightly acidic film on the surface of the skin has led to the development of acidic emollients for skin care. In this context, the effect of citric acid-coated textiles on atopic skin has not been examined to date. A textile carrier composed of cellulose fibres was coated with a citric acid surface layer by esterification, ensuring a constant pH of 5.5-6.5. Twenty patients with AE or atopic diathesis were enrolled in the study. In a double-blind, half-side experiment, patients had to wear these textiles for 12 h a day for 14 days. On day 0 (baseline), 7 and 14, tolerability (erythema, pruritus, eczema, wearing comfort) and efficacy on skin barrier were assessed by TEWL skin hydration (corneometry/capacitance), pH and clinical scoring of eczema (SCORAD). Citric acid-coated textiles were well tolerated and improved eczema and objective parameters of skin physiology, including barrier function and a reduced skin surface pH, with potential lower pathogenic microbial colonisation. PMID:24953993

  8. Intracellular pH regulation in rainbow trout (Oncorhynchus mykiss) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent.

    PubMed

    Tuominen, A; Rissanen, E; Bogdanova, A; Nikinmaa, M

    2003-06-01

    We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes. PMID:12820008

  9. Significance of pH on the Cytotoxic Potential of the Water Disinfection By-Product Iodoacetic Acid

    EPA Science Inventory

    Significance of pH on the Cytotoxic Potential of the Water Disinfection By-Product Iodoacetic Acid Vicki Richardson1, Susan D. Richardson2, Mary Moyer3, Jane Ellen Simmons1, and Anthony DeAngelo1, 1U.S. Environmental Protection Agency, Research Triangle Park, NC, 2University of...

  10. Directed Evolution of Metabolic Pathways in Microbial Populations. I. Modification of the Acid Phosphatase Ph Optimum in S. CEREVISIAE

    PubMed Central

    Francis, J. C.; Hansche, P. E.

    1972-01-01

    An experimental system for directing the evolution of enzymes and metabolic pathways in microbial populations is proposed and an initial test of its power is provided.—The test involved an attempt to genetically enhance certain functional properties of the enzyme acid phosphatase in S. cerevisiae by constructing an environment in which the functional changes desired would be "adaptive". Naturally occurring mutations in a population of 109 cells were automatically and continuously screened, over 1,000 generations, for their effect on the efficiency (Km) and activity of acid phosphatase at pH 6, and for their effect on the efficiency of orthophosphate metabolism.—The first adaptation observed, M1, was due to a single mutational event that effected a 30% increase in the efficiency of orthophosphate metabolism. The second, M2, effected an adaptive shift in the pH optimum of acid phosphatase and an increase in its activity over a wide range of pH values (an increment of 60% at pH 6). M2 was shown to result from a single mutational event in the region of the acid phosphatase structural gene. The third, M3, effected cell clumping, an adaptation to the culture apparatus that had no effect on phosphate metabolism.—The power of this system for directing the evolution of enzymes and of metabolic pathways is discussed in terms of the kinetic properties of the experimental system and in terms of the results obtained. PMID:4552227

  11. Direct Measurement of pH in Individual Particles via Raman Microspectroscopy and Variation in Acidity with Relative Humidity.

    PubMed

    Rindelaub, Joel D; Craig, Rebecca L; Nandy, Lucy; Bondy, Amy L; Dutcher, Cari S; Shepson, Paul B; Ault, Andrew P

    2016-02-18

    Atmospheric aerosol acidity is an important characteristic of aqueous particles, which has been linked to the formation of secondary organic aerosol by catalyzing reactions of oxidized organic compounds that have partitioned to the particle phase. However, aerosol acidity is difficult to measure and traditionally estimated using indirect methods or assumptions based on composition. Ongoing disagreements between experiments and thermodynamic models of particle acidity necessitate improved fundamental understanding of pH and ion behavior in high ionic strength atmospheric particles. Herein, Raman microspectroscopy was used to determine the pH of individual particles (H2SO4+MgSO4) based on sulfate and bisulfate concentrations determined from νs(SO4(2-)) and νs(HSO4(-)), the acid dissociation constant, and activity coefficients from extended Debye-Hückel calculations. Shifts in pH and peak positions of νs(SO4(2-)) and νs(HSO4(-)) were observed as a function of relative humidity. These results indicate the potential for direct spectroscopic determination of pH in individual particles and the need to improve fundamental understanding of ion behavior in atmospheric particles. PMID:26745214

  12. EFFECTS OF PH, SOLID/SOLUTION RATIO, IONIC STRENGTH, AND ORGANIC ACIDS ON PB AND CD SOPRTION ON KAOLINITE

    EPA Science Inventory

    Potentiometric and ion-selective electrode titrations together with batch sorption/desorption experiments, were performed to explain the aqueous and surface complexation reactions between kaolinite, Pb, Cd and three organic acids. Variables included pH, ionic strength, metal conc...

  13. Rat epididymal luminal fluid acid beta-D-galactosidase optimally hydrolyses glycoprotein substrate at neutral pH.

    PubMed Central

    Skudlarek, M D; Tulsiani, D R; Orgebin-Crist, M C

    1992-01-01

    Several glycosidases, purified and characterized from mammalian tissues, have been shown to be optimally active under acidic conditions when p-nitrophenyl (PNP) or 4-methylumbelliferyl glycosides are used as substrates. Although high levels of the glycosidases are present in the epididymal lumen, their physiological role remains uncertain. To be functional, the glycosidases are expected to be enzymatically active at or near the physiological pH of luminal fluid. In this report, we demonstrate that the rat epididymal luminal fluid beta-D-galactosidase, optimally active toward PNP beta-D-galactoside at pH 3.5, shows maximum activity towards a glycoprotein substrate ([Gal-3H]fetuin) at neutral pH. Several lines of evidence, including immunoprecipitation studies using antibody to the acid beta-D-galactosidase, and substrate competition studies, indicate that PNP galactosidase and [3H]Gal galactosidase activities are caused by a single enzyme, and that the two substrates are probably cleaved by the same catalytic site(s). Competition studies with various disaccharides indicate that this enzyme is capable of cleaving a variety of galactose linkages found in both O- and N-linked oligosaccharides. Molecular-sieve column chromatography of the beta-D-galactosidase of luminal fluid under several conditions of buffer and pH show that, whereas the enzyme eluted as a tetramer (apparent M(r) 320,000) under acidic conditions (pH 3.5-4.3), only dimers and monomers (apparent M(r) 180,000 and 92,000 respectively) were observed in neutral conditions (pH 6.8). This aggregation/dissociation phenomenon is reversible. These studies indicate that beta-D-galactosidase is present in the luminal fluid in dissociated forms, and is therefore optimally active towards glycoprotein substrates at physiological pH. The potential role of the enzyme in modification of sperm surface glycoproteins is discussed. PMID:1417750

  14. Denitrification potential in stream sediments impacted by acid mine drainage: effects of pH, various electron donors, and iron.

    PubMed

    Baeseman, J L; Smith, R L; Silverstein, J

    2006-02-01

    Acid mine drainage (AMD) contaminates thousands of kilometers of stream in the western United States. At the same time, nitrogen loading to many mountain watersheds is increasing because of atmospheric deposition of nitrate and increased human use. Relatively little is known about nitrogen cycling in acidic, heavy-metal-laden streams; however, it has been reported that one key process, denitrification, is inhibited under low pH conditions. The objective of this research was to investigate the capacity for denitrification in acidified streams. Denitrification potential was assessed in sediments from several Colorado AMD-impacted streams, ranging from pH 2.60 to 4.54, using microcosm incubations with fresh sediment. Added nitrate was immediately reduced to nitrogen gas without a lag period, indicating that denitrification enzymes were expressed and functional in these systems. First-order denitrification potential rate constants varied from 0.046 to 2.964 day(-1). The pH of the microcosm water increased between 0.23 and 1.49 pH units during denitrification. Additional microcosm studies were conducted to examine the effects of initial pH, various electron donors, and iron (added as ferrous and ferric iron). Decreasing initial pH decreased denitrification; however, increasing pH had little effect on denitrification rates. The addition of ferric and ferrous iron decreased observed denitrification potential rate constants. The addition of glucose and natural organic matter stimulated denitrification potential. The addition of hydrogen had little effect, however, and denitrification activity in the microcosms decreased after acetate addition. These results suggest that denitrification can occur in AMD streams, and if stimulated within the environment, denitrification might reduce acidity. PMID:16463131

  15. Denitrification potential in stream sediments impacted by acid mine drainage: Effects of pH, various electron donors, and iron

    USGS Publications Warehouse

    Baeseman, J.L.; Smith, R.L.; Silverstein, J.

    2006-01-01

    Acid mine drainage (AMD) contaminates thousands of kilometers of stream in the western United States. At the same time, nitrogen loading to many mountain watersheds is increasing because of atmospheric deposition of nitrate and increased human use. Relatively little is known about nitrogen cycling in acidic, heavy-metal-laden streams; however, it has been reported that one key process, denitrification, is inhibited under low pH conditions. The objective of this research was to investigate the capacity for denitrification in acidified streams. Denitrification potential was assessed in sediments from several Colorado AMD-impacted streams, ranging from pH 2.60 to 4.54, using microcosm incubations with fresh sediment. Added nitrate was immediately reduced to nitrogen gas without a lag period, indicating that denitrification enzymes were expressed and functional in these systems. First-order denitrification potential rate constants varied from 0.046 to 2.964 day-1. The pH of the microcosm water increased between 0.23 and 1.49 pH units during denitrification. Additional microcosm studies were conducted to examine the effects of initial pH, various electron donors, and iron (added as ferrous and ferric iron). Decreasing initial pH decreased denitrification; however, increasing pH had little effect on denitrification rates. The addition of ferric and ferrous iron decreased observed denitrification potential rate constants. The addition of glucose and natural organic matter stimulated denitrification potential. The addition of hydrogen had little effect, however, and denitrification activity in the microcosms decreased after acetate addition. These results suggest that denitrification can occur in AMD streams, and if stimulated within the environment, denitrification might reduce acidity. ?? Springer Science+Business Media, Inc. 2006.

  16. Bleb formation is induced by alkaline but not acidic pH in estrogen receptor silenced breast cancer cells.

    PubMed

    Khajah, Maitham A; Mathew, Princy M; Alam-Eldin, Nada S; Luqmani, Yunus A

    2015-04-01

    De novo and acquired resistance to endocrine-based therapies in breast cancer occurs in parallel with epithelial to mesenchymal transition (EMT), which is associated with enhanced proliferative and metastatic potential, and poor clinical outcome. We have established several endocrine insensitive breast cancer lines by shRNA-induced depletion of estrogen receptor (ER) by transfection of MCF7 cells. All of these exhibit EMT. We have previously reported that brief exposure of specifically ER- breast cancer cells, to extracellular alkaline pH, results in cell rounding and segregation, and leads to enhanced invasive potential. In this study we describe more detailed morphological changes and compare these with cell exposure to acidic pH. Morphological changes and localization of various molecules critical for cell adhesion and motility, associated with pH effects, were assessed by live cell microscopy, electron microscopy, and immunofluorescence. Exposure of either ER- or ER+ breast cancer cells to extracellular acidic pH did not induce significant changes in morphological appearance. Conversely, brief exposure of specifically ER silenced cells, to alkaline pH, resulted in cell contractolation and formation of bleb-like actin-rich structures which were evenly distributed on the outer membrane. Integrin α2, FAK, and JAM-1 were found in the cytoplasm streaming into the newly formed blebs. These blebs appear to be related to cell polarity and movement. Pre-treatment with cytochalasin-D or inhibitors of Rho or MLCK prevented both contractolation and bleb formation. Our data suggest that the effect of pH on the microenvironment of endocrine resistant breast cancer cells needs to be more extensively investigated. Alkaline, rather than acidic pH, appears to induce dramatic morphological changes, and enhances their invasive capabilities, through re-organization of cortical actin. PMID:25672508

  17. Addition of formic acid or starter cultures to liquid feed. Effect on pH, microflora composition, organic acid concentration and ammonia concentration.

    PubMed

    Canibe, N; Miquel, N; Miettinen, H; Jensen, B B

    2001-01-01

    Some of the charateristics of good quality fermented liquid feed (FLF) are low pH, high numbers of lactic acid bacteria, and low numbers of enterobacteria. In order to test strategies to avoid a proliferation of enterobacteria during the initial phase of FLF elaboration, two in vitro studies were carried out. Addition of various doses of formic acid or two different starter cultures were tested. Adding 0.1% formic acid or L. plantarum VTT E-78076 to the liquid feed seemed to be addecuate ways of inhibiting the growth of enterobacteria, without depleting the growth of lactic acid bacteria. PMID:15954629

  18. Human dental plaque pH, and the organic acid and free amino acid profiles in plaque fluid, after sucrose rinsing.

    PubMed

    Higham, S M; Edgar, W M

    1989-01-01

    The relationship between these factors was studied in plaque and plaque fluid samples taken at intervals during the Stephan pH curve following a sucrose mouth rinse. Levels of lactate rose after the rinse, then fell during the pH recovery phase. Levels of acetate, propionate and phosphate fell after rinsing, then rose again. Amino acid concentrations also changed, with many showing a fall followed by a rise; others rising then falling; and some showing a more variable or complex pattern. In resting plaque fluid, only alanine, proline, glutamic acid, glycine and ammonia were present at concentrations above 1 mmol/l. Delta-aminovaleric acid was detected at levels below those that have been found in monkeys. Hydroxyproline and hydroxylysine were consistently detected, levels of arginine were generally low, and those of cystine consistently very low. The results may provide a basis for understanding the complex metabolic interrelations that occur in the course of the Stephan curve and which may reflect or produce the observed pH changes. They suggest that besides the amount of acid produced, the type of acid, buffering power and base production should be considered as determinants of plaque pH. PMID:2597027

  19. Effect of the Fructus Ligustri Lucidi extract and its monomers quercetin and oleanolic acid on the adhesion and migration of melanocytes and intracellular actin

    PubMed Central

    WU, YANHUA; LI, QILIN; LI, XIANGJUN; HE, DANHUA; NIU, MU; LU, XIAOJUAN; LI, HUI

    2016-01-01

    The present study aimed to investigate the effects of the Fructus Ligustri Lucidi (FLL) extract and its monomers quercetin and oleanolic acid on the adhesion and migration of human epidermal melanocytes (MCs) and intracellular actin. The human epidermal MCs were cultured and identified. The cells were treated with different concentrations of FLL extract, quercetin and oleanolic acid. The adhesion and migration abilities of the cells were determined by the fibronectin-coated culture experiment and Transwell assay, respectively. The structure and distribution of intracellular actin were observed by confocal laser microscopy, with semi-quantitative analysis. Results showed that compared with the control group, 0.0375–0.3 mg/ml of the FLL extract and 40 µM quercetin significantly improved the adhesion rate of MCs (P<0.05). The numbers of MCs permeating the microporous membrane in the 0.15 mg/ml FLL extract and 12 µM oleanolic acid groups were 43.7 and 30.3, respectively, significantly higher compared to the control group (P<0.01). In the control group, the intracellular actin was less, and the stress fiber structure was not clear. In the 0.15 mg/ml FLL extract, 12 µM oleanolic acid and 40 µM quercetin groups, there were numerous bunched stress fibers, indicating the aggregation of filamentous fibrous actin. The mean optical densities of actin expression in the 0.15 mg/ml FLL extract, 12 µM oleanolic acid and 40 µM quercetin groups were significantly higher compared to the control group (P<0.05). The FLL extract has a significant stimulatory effect on the adhesion and migration of human epidermal MCs. The mechanism may be associated with the promotion of intracellular actin cytoskeleton aggregation. PMID:27123251

  20. Kinetic study of the reaction of sulfamethoxazole and glucose under acidic conditions: I. Effect of pH and temperature.

    PubMed

    Lucida, H; Parkin, J E; Sunderland, V B

    2000-07-20

    The kinetics of the reaction of sulfamethoxazole (SMX) in 5% w/v glucose to form the corresponding alpha- and beta-glucosylamines over the pH range of 0.80-6.88 at 37 degrees C has been investigated. The identity of the glucosylamines was determined by 1H-nuclear magnetic resonance spectroscopy of an authentic sample of the alpha-glucosylamine (USP) and the reaction products, and by interconversion of this compound to the corresponding beta-anomer. The reaction followed pseudo first-order reversible kinetics and involved specific acid and general acid-base catalysis. The pH-rate profile demonstrated that over the pH range of 0.80-2.90 and 5.50-6. 88 the reactions were dependent on H(+) concentration but pH independent between pH 3.00-5.45, which reflects the influence of ionization of SMX and the glucosylamines on the reversible reaction. Interpretation of the data with respect to kinetic models and rate equations for the formation and hydrolysis of the glucosylamines was investigated. Temperature dependence studies followed the Arrhenius equation with an Ea of 49.28 kJ mol(-1) for the forward and 63.46 kJ mol(-1) for the reverse reaction at pH 2.89 respectively. PMID:10915926

  1. A pH- and thermo-responsive poly(amino acid)-based drug delivery system.

    PubMed

    Liu, Na; Li, Bingqiang; Gong, Chu; Liu, Yuan; Wang, Yanming; Wu, Guolin

    2015-12-01

    A pH- and thermo-responsive poly(amino acid)-based amphiphilic copolymer was developed, functioning as a tumour targeting drug delivery system with good biocompatibility and biodegradability. To provide multi-stimuli sensitivity characteristics to the poly(amino acid)s, the polyaspartamide scaffold has been functionalized with N,N-diisopropylamide groups via aminolysis reaction of polysuccinimide. PEG chains have also been chemically grafted to the poly(amino acid) backbone through acid-labile hydrazone linkages, providing a removable shield for the poly(amino acid) based nanoparticles. Furthermore, doxorubicin was chemically linked to the copolymer chain via hydrazone bonds, acting as the hydrophobic moiety to drive the polymeric self-assembly. Free doxorubicin molecules could be encapsulated into the self-assembled nanoparticles via hydrophobic interactions and molecular π-π stacking. The results obtained show that the drug release can be triggered by the temperature with a significantly increased release being observed under acidic conditions. The cytotoxicity behaviour of the copolymers and drug-loaded nanoparticles was investigated in vitro at varying pH values and different temperatures. In doing so, superior characteristics concerning compatibility and anti-cancer activity could be observed. PMID:26454546

  2. [Effects of solution pH and simulated acid rain on the behavior of two sulfonylurea herbicides in soil].

    PubMed

    Zhang, Wei; Wang, Jin-Jun

    2007-03-01

    By the methods of batch equilibration and leaching, this paper studied the effects of solution pH and simulated acid rain on the behavior of bensulfuron-methyl and metsulfuron-methyl in soil. The results showed that the adsorption isotherms of these two herbicides fitted Freundlich equation well, and their adsorbed amounts reduced obviously with the increasing pH of water-soil system, which in turn promoted the translocation of the herbicides in soil. The adsorption coefficient (Kf) was positively correlated with soil organic matter and clay contents, while negatively correlated with soil pH. The higher pH of simulated acid rain had a greater contribution on the leaching of the two sulfonylurea herbicides, and their leached amount was increased with increasing acid rain. There was a close relationship between the leaching of the herbicides and the properties of soil. The soils with higher contents of organic matter and clay had a greater retention capability to the herbicides. PMID:17552202

  3. Relative effectiveness of various anions on the solubility of acidic Hypoderma lineatum collagenase at pH 7.2.

    PubMed Central

    Carbonnaux, C.; Ries-Kautt, M.; Ducruix, A.

    1995-01-01

    The effects of various anions on decreasing the solubility of acidic Hypoderma lineatum collagenase at pH 7.2 and 18 degrees C were qualitatively defined by replacing the crystallizing agent of known crystallization conditions by various ammonium salts. The solubility curves measured in the presence of the sulfate, phosphate, citrate, and chloride ammonium salts gave the following ranking of anions: HPO4(2-)/H2PO4- > SO4(2-) > citrate 3-/citrate2- >> Cl-. This order is in agreement with the Hofmeister series. In a previous study on the solubility at pH 4.5 of lysozyme, a basic protein, the effectiveness of anions in decreasing the solubility was found to be in the reverse order. This suggests that the effectiveness of anions in the crystallization of proteins is dependent on the net charge of the protein, i.e., depending on whether a basic protein is crystallized at acidic pH or an acidic protein at basic pH. PMID:8535249

  4. Effect of L-lactic acid, short-chain fatty acids, and pH in cecal infusate on morphometric and cell kinetic parameters of rat cecum.

    PubMed

    Ichikawa, H; Sakata, T

    1997-08-01

    We studied the influences of cecal infusion of NaCl, short-chain fatty acids (SCFA), and L-lactic acid at pH 5.0 or 7.0 for seven days on morphometric and cell kinetic parameters of the rat cecum. SCFA increased relative weight of the mucosa and submucosa, crypt size, and mitotic index in the cecum. L-Lactic acid stimulated mitosis only at pH 5.0. Crypt size correlated positively to epithelial proliferative activity only when NaCl or L-lactic acid was infused. SCFA should have changed the balance between production and loss of the cecal epithelial cells. The infusate pH by itself had no effect, but modified the effects of SCFA and L-lactic acid in different ways. Crypt size correlated positively to the logarithm of daily proton load of infusates. The above results indicate that epithelial cell proliferation in the cecum is influenced by both SCFA and L-lactic acid, although differently, and by proton load. PMID:9286223

  5. Demonstration of in situ product recovery of butyric acid via CO2 -facilitated pH swings and medium development in two-phase partitioning bioreactors.

    PubMed

    Peterson, Eric C; Daugulis, Andrew J

    2014-03-01

    Production of organic acids in solid-liquid two-phase partitioning bioreactors (TPPBs) is challenging, and highly pH-dependent, as cell growth occurs near neutral pH, while acid sorption occurs only at low pH conditions. CO2 sparging was used to achieve acidic pH swings, facilitating undissociated organic acid uptake without generating osmotic stress inherent in traditional acid/base pH control. A modified cultivation medium was formulated to permit greater pH reduction by CO2 sparging (pH 4.8) compared to typical media (pH 5.3), while still possessing adequate nutrients for extensive cell growth. In situ product recovery (ISPR) of butyric acid (pKa = 4.8) produced by Clostridium tyrobutyricum was achieved through intermittent CO2 sparging while recycling reactor contents through a column packed with absorptive polymer Hytrel® 3078. This polymer was selected on the basis of its composition as a polyether copolymer, and the use of solubility parameters for predicting solute polymer affinity, and was found to have a partition coefficient for butyric acid of 3. Total polymeric extraction of 3.2 g butyric acid with no CO2 mediated pH swings was increased to 4.5 g via CO2 -facilitated pH shifting, despite the buffering capacity of butyric acid, which resists pH shifting. This work shows that CO2 -mediated pH swings have an observable positive effect on organic acid extraction, with improvements well over 150% under optimal conditions in early stage fermentation compared to CO2 -free controls, and this technique can be applied other organic acid fermentations to achieve or improve ISPR. PMID:23996152

  6. Urea Fertilizer and pH Influence on Sorption Process of Flumetsulam and MCPA Acidic Herbicides in a Volcanic Soil.

    PubMed

    Palma, Graciela; Jorquera, Milko; Demanet, Rolando; Elgueta, Sebastian; Briceño, Gabriela; de la Luz Mora, María

    2016-01-01

    The aim of this study was to evaluate the influence of urea fertilizer and pH on the sorption process of two acidic herbicides, flumetsulam (2',6'-difluoro-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonanilide) and MCPA (4-chloro--tolyloxyacetic acid), on an Andisol. Urea reduced the adsorption of MCPA but not that of flumetsulam. The Freundlich parameter of MCPA decreased from 8.5 to 5.1 mg L kg. This finding could be attributed to an increase in dissolved organic C due to an initial increase in soil pH for urea application. The higher acidic character of MCPA compared with that of flumetsulam produced a greater hydrolysis of urea, leading to a further pH increase. A marked effect of pH on the adsorption of both herbicides was observed. The organic C distribution coefficient () values for flumetsulam were in the range of 74 to 10 L kg, while those of MCPA were in the range of 208 to 45 L kg. In the kinetic studies, the pseudo-second-order model appeared to fit the data best ( > 0.994). The initial adsorption rates () ranged from 20.00 to 4.59 mg kg h for flumetsulam and from 125.00 to 25.60 mg kg hfor MCPA. Both herbicides were adsorbed rapidly during the first stage of the sorption process, and the rates of sorption were dependent on pH. The application of the Elovich and Weber-Morris models led us to conclude that mass transfer through the boundary layer and, to a lesser degree, intraparticle diffusion were influenced by the chemical character of the herbicide. These results suggest that urea application could increase leaching of acid herbicides in soils. PMID:26828188

  7. Controlling the pH of acid cheese whey in a two-stage anaerobic digester with sodium hydroxide

    SciTech Connect

    Ghaly, A.E.; Ramkumar, D.R.

    1999-07-01

    Anaerobic digestion of cheese whey offers a two-fold benefit: pollution potential reduction and biogas production. The biogas, as an energy source, could be used to reduce the consumption of traditional fuels in the cheese plant. However, as a result of little or no buffering capacity of whey, the pH of the anaerobic digester drops drastically and the process is inhibited. In this study, the effect of controlling the pH of the second chamber of a two-stage, 150 L anaerobic digester operating on cheese whey on the quality and quantity of biogas and the pollution potential reduction, was investigated using sodium hydroxide. The digester was operated at a temperature of 35 C and a hydraulic retention time of 15 days for three runs (no pH control, pH control with no reseeding, and ph control with reseeding) each lasting 50 days. The results indicated that operating the digester without pH control resulted in a low pH (3.3) which inhibited the methanogenic bacteria. The inhibition was irreversible and the digester did not recover (no methane production) when the pH was restored to 7.0 without reseeding, as the observed increased gas production was a false indication of recovery because the gas was mainly carbon dioxide. The addition of base resulted in a total alkalinity of 12,000 mg/L as CaCO{sub 3}. When the system was reseeded and the pH controlled, the total volatile acid concentration was 15,100 mg/L (as acetic acid), with acetic (28%), propionic (21%), butyric (25%), valeric (8%), and caproic (15%) acids as the major constituents. The biogas production was 62.6 L/d (0.84 m{sup 3}/m{sup 3}/d) and the methane content was 60.7%. Reductions of 27.3, 30.4 and 23.3% in the total solids, chemical oxygen demand and total kjeldahl nitrogen were obtained, respectively. The ammonium nitrogen content increased significantly (140%).

  8. Potentiometric pH Measurements of Acidity Are Approximations, Some More Useful than Others

    ERIC Educational Resources Information Center

    de Levie, Robert

    2010-01-01

    A recent article by McCarty and Vitz "demonstrating that it is not true that pH = -log[H+]" is examined critically. Then, the focus shifts to underlying problems with the IUPAC definition of pH. It is shown how the potentiometric method can provide "estimates" of both the IUPAC-defined hydrogen activity "and" the hydrogen ion concentration, using…

  9. Sulfur mustard-induced increase in intracellular free calcium level and arachidonic acid release from cell membrane

    SciTech Connect

    Ray, R.; Legere, R.H.; Majerus, B.J.; Petrali, J.P.

    1995-12-31

    The mechanism of action of the alkylating agent bis-(2-chloroethyl)sulfide (sulfur mustard, SM) was studied using the in thai vitro mouse neuroblastoma-rat glioma hybrid NG 108-1 S clonal p cell line model. Following 0.3 mM SM exposure, cell viability remained high (>80% of untreated control) up to 9 hr and then declined steadily to about 40% of control after 20-24 hr. During the early period of SM exposure, when there was no significant cell viability loss, the following effects were observed. The cellular glutathione level decreased 20% after 1 hr and 34% after 6 hr. Between 2 and 6 hr, there was a time-dependent increase (about 10 to 30%) in intracellular free calcium (Ca2+), which was localized to the limiting membrane of swollen endoplasmic reticula and mitochondria, to euchromatin areas of the nucleus, and to areas of the cytosol and plasma membrane. Moreover,there was also a time-dependent increase in the release of isotopically labeled arachidonic acid ((3H)AA) from cellular membranes. Increase in (3H)AA release was 28% at 3 hr and about 60-80% between 6 and 9 hr. This increase in I3HIAA release was inhibited by quinacrine (20 uM), which is a phospholipase (PLA2) inhibitor. At 16 hr after SM exposure, there was a large increase (about 200% of control) in I3HIAA release, which was coincident with a 50% loss of cell viability. These results suggest a Ca2+-mediated toxic mechanism of SM via PLA2 activation and arachidonate release.

  10. Bile acids and pH values in total feces and in fecal water from habitually omnivorous and vegetarian subjects.

    PubMed

    van Faassen, A; Hazen, M J; van den Brandt, P A; van den Bogaard, A E; Hermus, R J; Janknegt, R A

    1993-12-01

    Twenty habitually omnivorous subjects and 19 habitually lactoovovegetarian subjects aged 59-65 y collected feces during 4 consecutive days. The concentrations of bile acids in total feces did not differ between the omnivores and vegetarians, but the bile acid concentrations in fecal water were significantly lower in the vegetarians. The concentration of the colorectal cancer-predicting bile acid deoxycholic acid in fecal water was explained by the intake of saturated fat and the daily fecal wet weight (r2 = 0.50). Fecal pH did not differ between the omnivores and vegetarians. This variable was significantly (P < 0.05) explained by the intake of calcium (r2 = 0.30); 24-h fecal wet weight and defecation frequency were significantly higher in the vegetarians. In conclusion, our vegetarian subjects had a lower concentration of deoxycholic acid in fecal water, higher fecal wet weight, and higher defecation frequency than the omnivorous subjects. PMID:8249879

  11. Dissolution kinetics of a lunar glass simulant at 25 degrees C: the effect of pH and organic acids

    NASA Technical Reports Server (NTRS)

    Eick, M. J.; Grossl, P. R.; Golden, D. C.; Sparks, D. L.; Ming, D. W.

    1996-01-01

    The dissolution kinetics of a simulated lunar glass were examined at pH 3, 5, and 7. Additionally, the pH 7 experiments were conducted in the presence of citric and oxalic acid at concentrations of 2 and 20 mM. The organic acids were buffered at pH 7 to examine the effect of each molecule in their dissociated form. At pH 3, 5, and 7, the dissolution of the synthetic lunar glass was observed to proceed via a two-stage process. The first stage involved the parabolic release of Ca, Mg, Al, and Fe, and the linear release of Si. Dissolution was incongruent, creating a leached layer rich in Si and Ti which was verified by transmission electron microscopy (TEM). During the second stage the release of Ca, Mg, Al, and Fe was linear. A coupled diffusion/surface dissolution model was proposed for dissolution of the simulated lunar glass at pH 3, 5, and 7. During the first stage the initial release of mobile cations (i.e., Ca, Mg, Al, Fe) was limited by diffusion through the surface leached layer of the glass (parabolic release), while Si release was controlled by the hydrolysis of the Si-O-Al bonds at the glass surface (linear release). As dissolution continued, the mobile cations diffused from greater depths within the glass surface. A steady-state was then reached where the diffusion rate across the increased path lengths equalled the Si release rate from the surface. In the presence of the organic acids, the dissolution of the synthetic lunar glass proceeded by a one stage process. The release of Ca, Mg, Al, and Fe followed a parabolic relationship, while the release of Si was linear. The relative reactivity of the organic acids used in the experiments was citrate > oxalate. A thinner leached layer rich in Si/Ti, as compared to the pH experiments, was observed using TEM. Rate data suggest that the chemisorption of the organic anion to the surface silanol groups was responsible for enhanced dissolution in the presence of the organic acids. It is proposed that the increased

  12. The pH low insertion peptide pHLIP Variant 3 as a novel marker of acidic malignant lesions

    PubMed Central

    Tapmeier, Thomas T.; Moshnikova, Anna; Beech, John; Allen, Danny; Kinchesh, Paul; Smart, Sean; Harris, Adrian; McIntyre, Alan; Engelman, Donald M.; Andreev, Oleg A.; Reshetnyak, Yana K.; Muschel, Ruth J.

    2015-01-01

    Current strategies for early detection of breast and other cancers are limited in part because some lesions identified as potentially malignant do not develop into aggressive tumors. Acid pH has been suggested as a key characteristic of aggressive tumors that might distinguish aggressive lesions from more indolent pathology. We therefore investigated the novel class of molecules, pH low insertion peptides (pHLIPs), as markers of low pH in tumor allografts and of malignant lesions in a mouse model of spontaneous breast cancer, BALB/neu-T. pHLIP Variant 3 (Var3) conjugated with fluorescent Alexa546 was shown to insert into tumor spheroids in a sequence-specific manner. Its signal reflected pH in murine tumors. It was induced by carbonic anhydrase IX (CAIX) overexpression and inhibited by acetazolamide (AZA) administration. By using 31P magnetic resonance spectroscopy (MRS), we demonstrated that pHLIP Var3 was retained in tumors of pH equal to or less than 6.7 but not in tissues of higher pH. In BALB/neu-T mice at different stages of the disease, the fluorescent signal from pHLIP Var3 marked cancerous lesions with a very low false-positive rate. However, only ∼60% of the smallest lesions retained a pHLIP Var3 signal, suggesting heterogeneity in pH. Taken together, these results show that pHLIP can identify regions of lower pH, allowing for its development as a theranostic tool for clinical applications. PMID:26195776

  13. Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids

    PubMed Central

    Liang, Wanling; Kwok, Philip C.L.; Chow, Michael Y.T.; Tang, Patricia; Mason, A. James; Chan, Hak-Kim; Lam, Jenny. K.W.

    2013-01-01

    Nucleic acids have the potential to be used as therapies or vaccines for many different types of disease but delivery remains the most significant challenge to their clinical adoption. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar fluid (BALF), making this class of peptides attractive candidates for delivering nucleic acids to lung tissues. To further assess the suitability of pH responsive peptides for pulmonary delivery by inhalation, dry powder formulations of pH responsive peptides and plasmid DNA, with mannitol as carrier, were produced by either spray drying (SD) or spray freeze drying (SFD). The properties of the two types of powders were characterised and compared using scanning electron microscopy (SEM), next generation impaction (NGI), gel retardation and in vitro transfection via a twin-stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). Although the aerodynamic performance and transfection efficacy of both powders were good, the overall performance revealed SD powders to have a number of advantages over SFD powders and are the more effective formulation with potential for efficient nucleic acid delivery through inhalation. PMID:23702276

  14. [The effect of pH and amount of antacids on bile acid binding in a quasi-natural reflux milieu].

    PubMed

    Kurtz, W; Güldütuna, S; Leuschner, U

    1991-05-01

    Bile acid adsorption may be one therapeutical mechanism of antacids. Little is known about the effect of pH and amount of antacid on bile acid adsorption. Therefore we carried out the following investigations using a lattice [correction of lettuce] layer antacid as a model substance. 5 ml of "quasi-natural reflux milieu" were mixed with 0.5, 1 or 2 ml of hydrotalcite and adjusted to pH 3, 5 or 7. The highest total bile acid adsorption was found at pH 3, the degree of bile acid adsorption correlated with bile acid lipophilicity, i.e. the most lipophilic and toxic bile acids are adsorbed best. High adsorption of lipophilic and particularly toxic bile acids even at low gastric pH may help to explain the good therapeutic effect of low-dose antacids in gastric ulcer. PMID:1950032

  15. Cluster of Differentiation 38 (CD38) Mediates Bile Acid-induced Acinar Cell Injury and Pancreatitis through Cyclic ADP-ribose and Intracellular Calcium Release*

    PubMed Central

    Orabi, Abrahim I.; Muili, Kamaldeen A.; Javed, Tanveer A.; Jin, Shunqian; Jayaraman, Thottala; Lund, Frances E.; Husain, Sohail Z.

    2013-01-01

    Aberrant Ca2+ signals within pancreatic acinar cells are an early and critical feature in acute pancreatitis, yet it is unclear how these signals are generated. An important mediator of the aberrant Ca2+ signals due to bile acid exposure is the intracellular Ca2+ channel ryanodine receptor. One putative activator of the ryanodine receptor is the nucleotide second messenger cyclic ADP-ribose (cADPR), which is generated by an ectoenzyme ADP-ribosyl cyclase, CD38. In this study, we examined the role of CD38 and cADPR in acinar cell Ca2+ signals and acinar injury due to bile acids using pharmacologic inhibitors of CD38 and cADPR as well as mice deficient in Cd38 (Cd38−/−). Cytosolic Ca2+ signals were imaged using live time-lapse confocal microscopy in freshly isolated mouse acinar cells during perifusion with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 μm). To focus on intracellular Ca2+ release and to specifically exclude Ca2+ influx, cells were perifused in Ca2+-free medium. Cell injury was assessed by lactate dehydrogenase leakage and propidium iodide uptake. Pretreatment with either nicotinamide (20 mm) or the cADPR antagonist 8-Br-cADPR (30 μm) abrogated TLCS-induced Ca2+ signals and cell injury. TLCS-induced Ca2+ release and cell injury were reduced by 30 and 95%, respectively, in Cd38-deficient acinar cells compared with wild-type cells (p < 0.05). Cd38-deficient mice were protected against a model of bile acid infusion pancreatitis. In summary, these data indicate that CD38-cADPR mediates bile acid-induced pancreatitis and acinar cell injury through aberrant intracellular Ca2+ signaling. PMID:23940051

  16. Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions

    PubMed Central

    Kang, Bok Eum; Baker, Bradley J.

    2016-01-01

    An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response. PMID:27040905

  17. Pado, a fluorescent protein with proton channel activity can optically monitor membrane potential, intracellular pH, and map gap junctions.

    PubMed

    Kang, Bok Eum; Baker, Bradley J

    2016-01-01

    An in silico search strategy was developed to identify potential voltage-sensing domains (VSD) for the development of genetically encoded voltage indicators (GEVIs). Using a conserved charge distribution in the S2 α-helix, a single in silico search yielded most voltage-sensing proteins including voltage-gated potassium channels, voltage-gated calcium channels, voltage-gated sodium channels, voltage-gated proton channels, and voltage-sensing phosphatases from organisms ranging from mammals to bacteria and plants. A GEVI utilizing the VSD from a voltage-gated proton channel identified from that search was able to optically report changes in membrane potential. In addition this sensor was capable of manipulating the internal pH while simultaneously reporting that change optically since it maintains the voltage-gated proton channel activity of the VSD. Biophysical characterization of this GEVI, Pado, demonstrated that the voltage-dependent signal was distinct from the pH-dependent signal and was dependent on the movement of the S4 α-helix. Further investigation into the mechanism of the voltage-dependent optical signal revealed that inhibiting the dimerization of the fluorescent protein greatly reduced the optical signal. Dimerization of the FP thereby enabled the movement of the S4 α-helix to mediate a fluorescent response. PMID:27040905

  18. Thermodynamic modelling of the effect of hydroxycarboxylic acids on the solubility of plutonium at high pH

    SciTech Connect

    Moreton, A.D.

    1993-12-31

    A number of the hydroxycarboxlyic acids generated by the alkaline degradation of cellulosic wastes under reducing conditions in a cementitious repository can significantly increase the solubility of the actinides at high pH, especially plutonium. The solubility of plutonium at pH 12, in the presence of a range of hydroxycarboxylic acids containing a number of hydroxyl groups and between one and three carboxylate groups, has been modelled using the HARPHRQ code. All the plutonium-organic complexes assumed in the model are based on a stable unit in which a central plutonium ion is bound by four oxygen atoms. The oxygen atoms can be provided either by a deprotonated hydroxyl group on one of the ligands, or by hydroxide ions.

  19. pH dependence of iron photoreduction in a rocky mountain stream affected by acid mine drainage

    USGS Publications Warehouse

    McKnight, Diane M.; Kimball, B.A.; Runkel, R.L.

    2001-01-01

    The redox speciation of dissolved iron and the transport of iron in acidic, metal-enriched streams is controlled by precipitation and dissolution of iron hydroxides, by photoreduction of dissolved ferric iron and hydrous iron oxides, and by oxidation of the resulting dissolved ferrous iron. We examined the pH dependence of these processes in an acidic mine-drainage stream, St Kevin Gulch, Colorado, by experimentally increasing the pH of the stream from about 4.0 to 6.5 and following the downstream changes in iron species. We used a solute transport model with variable flow to evaluate biogeochemical processes controlling downstream transport. We found that at pH 6.4 there was a rapid and large initial loss of ferrous iron concurrent with the precipitation of aluminium hydroxide. Below this reach, ferrous iron was conservative during the morning but there was a net downstream loss of ferrous iron around noon and in the afternoon. Calculation of net oxidation rates shows that the noontime loss rate was generally much faster than rates for the ferrous iron oxidation at pH 6 predicted by Singer and Stumm (1970. Science 167: 1121). The maintenance of ferrous iron concentrations in the morning is explained by the photoreduction of photoreactive ferric species, which are then depleted by noon. Copyright ?? 2001 John Wiley & Sons, Ltd.

  20. pH dependence of iron photoreduction in a rocky mountain stream affected by acid mine drainage

    NASA Astrophysics Data System (ADS)

    McKnight, Diane M.; Kimball, Briant A.; Runkel, Robert L.

    2001-07-01

    The redox speciation of dissolved iron and the transport of iron in acidic, metal-enriched streams is controlled by precipitation and dissolution of iron hydroxides, by photoreduction of dissolved ferric iron and hydrous iron oxides, and by oxidation of the resulting dissolved ferrous iron. We examined the pH dependence of these processes in an acidic mine-drainage stream, St Kevin Gulch, Colorado, by experimentally increasing the pH of the stream from about 4·0 to 6·5 and following the downstream changes in iron species. We used a solute transport model with variable flow to evaluate biogeochemical processes controlling downstream transport. We found that at pH 6·4 there was a rapid and large initial loss of ferrous iron concurrent with the precipitation of aluminium hydroxide. Below this reach, ferrous iron was conservative during the morning but there was a net downstream loss of ferrous iron around noon and in the afternoon. Calculation of net oxidation rates shows that the noontime loss rate was generally much faster than rates for the ferrous iron oxidation at pH 6 predicted by Singer and Stumm (1970. Science 167: 1121). The maintenance of ferrous iron concentrations in the morning is explained by the photoreduction of photoreactive ferric species, which are then depleted by noon.

  1. Influence of levels of information as presented by different technologies on students' understanding of acid, base, and ph concepts

    NASA Astrophysics Data System (ADS)

    Nakhleh, Mary B.; Krajcik, Joseph S.

    We investigated how different levels of information presented by various technologies affected secondary students' understanding of acid, base, and pH concepts. Secondary students who were selected for the study had just completed their study of acid-base chemistry. No attempt was made to provide further instruction. We analyzed changes in the understanding of individual students by constructing concept maps from the propositions that the students used in interviews conducted before and after a series of acid-base titrations. After the initial interview, students were divided into three groups. Within each group, students individually performed the same set of titrations using different technologies: chemical indicators, pH meters, and microcomputer-based laboratories (MBL). After the titrations were completed, all students were interviewed again. We found that students using MBL exhibited a larger positive shift in their concept map scores, which indicates a greater differentiation and integration of their knowledge of acids and bases. The chemical indicator students exhibited a more moderate positive shift in their concept map scores, and the pH meter students exhibited a smaller positive shift. We also found that the MBL students constructed more inappropriate links in their concept maps than the chemical indicator or pH meter students. However, we speculate that this increased number of inappropriate links indicates a high level of involvement with the technology. We therefore argue that the level of information offered by the technology affected students' understanding of the chemical concepts.Received: 24 February 1993; Revised: 21 February 1994;

  2. Control of diapause by acidic pH and ammonium accumulation in the hemolymph of Antarctic copepods.

    PubMed

    Schründer, Sabine; Schnack-Schiel, Sigrid B; Auel, Holger; Sartoris, Franz Josef

    2013-01-01

    Life-cycles of polar herbivorous copepods are characterised by seasonal/ontogenetic vertical migrations and diapause to survive periods of food shortage during the long winter season. However, the triggers of vertical migration and diapause are still far from being understood. In this study, we test the hypothesis that acidic pH and the accumulation of ammonium (NH4 (+)) in the hemolymph contribute to the control of diapause in certain Antarctic copepod species. In a recent study, it was already hypothesized that the replacement of heavy ions by ammonium is necessary for diapausing copepods to achieve neutral buoyancy at overwintering depth. The current article extends the hypothesis of ammonium-aided buoyancy by highlighting recent findings of low pH values in the hemolymph of diapausing copepods with elevated ammonium concentrations. Since ammonia (NH3) is toxic to most organisms, a low hemolymph pH is required to maintain ammonium in the less toxic ionized form (NH4 (+)). Recognizing that low pH values are a relevant factor reducing metabolic rate in other marine invertebrates, the low pH values found in overwintering copepods might not only be a precondition for ammonium accumulation, but in addition, it may insure metabolic depression throughout diapause. PMID:24143238

  3. A new hyaluronic acid pH sensitive derivative obtained by ATRP for potential oral administration of proteins.

    PubMed

    Fiorica, Calogero; Pitarresi, Giovanna; Palumbo, Fabio Salvatore; Di Stefano, Mauro; Calascibetta, Filippo; Giammona, Gaetano

    2013-11-30

    Atom transfer radical polymerization (ATRP) has been successfully employed to obtain a new derivative of hyaluronic acid (HA) able to change its solubility as a function of external pH and then to be potentially useful for intestinal release of bioactive molecules, included enzymes and proteins. In particular, a macroinitiator has been prepared by linking 2-bromo-2-methypropionic acid (BMP) to the amino groups of ethylenediamino derivative of tetrabutyl ammonium salt of HA (HA-TBA-EDA). This macroinititor, named HA-TBA-EDA-BMP has been used for the ATRP of sodium methacrylate (MANa) using a complex of Cu(I) and 2,2'-bipyridyl (Byp) as a catalyst. The resulting copolymer, named HA-EDA-BMP-MANa, has been characterized by (1)H NMR and size exclusion chromatography (SEC) analyses. A turbidimetric analysis has showed its pH sensitive behavior, being insoluble in simulated gastric fluid but soluble when pH increases more than 2.5. To confirm the ability of HA-EDA-BMP-MANa in protecting peptides or proteins from denaturation in acidic medium, α-chymotrypsin has been chosen as a model of protein molecule and its activity has been evaluated after entrapment into HA-EDA-BMP-MANa chains and treatment under simulated gastric conditions. Finally, cell compatibility has been evaluated by performing a MTS assay on murine dermal fibroblasts cultured with HA-EDA-BMP-MANa solutions. PMID:24060369

  4. Enhanced α-ketoglutaric acid production and recovery in Yarrowia lipolytica yeast by effective pH controlling.

    PubMed

    Morgunov, Igor G; Kamzolova, Svetlana V; Samoilenko, Vladimir A

    2013-10-01

    The replacement of chemical synthesis by environmentally friendly energy-efficient technologies for production of valuable metabolites is a principal strategy of developing biotechnological industry all over the world. In the present study, we develop a method for α-ketoglutaric acid (KGA) production from rapeseed oil with the use of Yarrowia lipolytica yeast. Sixty strains of Y. lipolytica yeasts were tested for their ability to produce KGA, and the strain Y. lipolytica 212 (Y. lipolytica VKM Y-2412) was selected as a promising KGA producer. Using a three-stage pH controlling, in which pH was 4.5 in the growth phase, then since 72 to 144 h, pH was maintained at 3.5 and in the later phase of acid production, the titration by KOH was switch off, selected strain produced 106.5 g l(-1) of KGA with mass yield of 0.95 g g(-1). KGA in the form of monopotassium salt was isolated from the culture broth and purified. The isolation procedure involved separation of biomass, extraction of residual triglycerides, filtrate bleaching, and acidification with mineral acid (to pH 2.8-3.4), concentration, precipitation of mineral salts, and crystallization of the product. The purity of KGA isolated from the culture filtrate reached 99.1 %. PMID:23948727

  5. pH induced dual "OFF-ON-OFF" switch: influence of a suitably placed carboxylic acid.

    PubMed

    Sadhu, Kalyan K; Mizukami, Shin; Yoshimura, Akimasa; Kikuchi, Kazuya

    2013-01-28

    The design and synthesis of molecular probes competent for pH signaling within or beyond a certain range is a complicated matter. Herein a new mechanism for ''OFF-ON-OFF'' absorbance and fluorescence intensities vs. pH behaviour is described. The probe design is based on the connection of carboxylic acid derivatized benzoxazole and 7-hydroxycoumarin/iminocoumarin parts. The protonation/deprotonation of the carboxylic acid (-COOH), N atom of benzoxazole ring and hydroxy part of the coumarin ring have been used for this mechanistic study. We have designed the molecule in such a fashion that deprotonation of the hydroxy part takes place at a lower pK(a) compared to deprotonation of the -COOH. The dual ''OFF-ON-OFF'' properties of our probes depend on the C-C bond between the two different heterocyclic parts. Quantum mechanical calculations showed that the particular 'C-C' bond has an additional π-character. The twisting around this bond in different forms is responsible for such an ''OFF-ON-OFF'' property. This mechanism is new in fluorescence alteration processes. The delocalization of charge from one heterocyclic part to the other heterocyclic part in the mono- and dianionic forms controls the ''OFF-ON-OFF'' properties. The role of the carboxylic acid group was examined using an acetyl substituted derivative. One of our probes was successfully applied in live cell imaging studies in media at different pH. PMID:23060072

  6. The PH gene determines fruit acidity and contributes to the evolution of sweet melons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acids are one of the three major components of fleshy fruit taste, together with sugars and volatile flavor compounds. However, the molecular-genetic control of acid accumulation in fruit is poorly understood and, to date, no genes responsible for acid accumulation in fleshy fruit have been function...

  7. The intracellular parasite Toxoplasma gondii depends on the synthesis of long chain and very long-chain unsaturated fatty acids not supplied by the host cell

    PubMed Central

    Ramakrishnan, Srinivasan; Docampo, Melissa D.; MacRae, James I.; Ralton, Julie E.; Rupasinghe, Thusitha; McConville, Malcolm J.; Striepen, Boris

    2015-01-01

    SUMMARY Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In T. gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here we demonstrate that the ER-localized fatty acid elongation (ELO) is essential for parasite growth. Conditional knock-down of the non-redundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. 13C-glucose and 13C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was by-passed by supplementing the media with specific fatty acids, indicating active, but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway. PMID:25825226

  8. Adaptive responses of Bacillus cereus ATCC14579 cells upon exposure to acid conditions involve ATPase activity to maintain their internal pH

    PubMed Central

    Senouci-Rezkallah, Khadidja; Jobin, Michel P; Schmitt, Philippe

    2015-01-01

    This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe) 7.0 or 5.5 and at a growth rate of 0.2 h−1. Population reduction and internal pH (pHi) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N’-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells. PMID:25740257

  9. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  10. Impact of temperature, pH, and salinity changes on the physico-chemical properties of model naphthenic acids.

    PubMed

    Celsie, Alena; Parnis, J Mark; Mackay, Donald

    2016-03-01

    The effects of temperature, pH, and salinity change on naphthenic acids (NAs) present in oil-sands process wastewater were modeled for 55 representative NAs. COSMO-RS was used to estimate octanol-water (KOW) and octanol-air (KOA) partition ratios and Henry's law constants (H). Validation with experimental carboxylic acid data yielded log KOW and log H RMS errors of 0.45 and 0.55 respectively. Calculations of log KOW, (or log D, for pH-dependence), log KOA and log H (or log HD, for pH-dependence) were made for model NAs between -20 °C and 40 °C, pH between 0 and 14, and salinity between 0 and 3 g NaCl L(-1). Temperature increase by 60 °C resulted in 3-5 log unit increase in H and a similar magnitude decrease in KOA. pH increase above the NA pKa resulted in a dramatic decrease in both log D and log HD. Salinity increase over the 0-3 g NaCl L(-1) range resulted in a 0.3 log unit increase on average for KOW and H values. Log KOW values of the sodium salt and anion of the conjugate base were also estimated to examine their potential for contribution to the overall partitioning of NAs. Sodium salts and anions of naphthenic acids are predicted to have on average 4 log units and 6 log units lower log KOW values, respectively, with respect to the corresponding neutral NA. Partitioning properties are profoundly influenced by the by the relative prevailing pH and the substance's pKa at the relevant temperature. PMID:26706930

  11. In situ measurement of reaction volume and calculation of pH of weak acid buffer solutions under high pressure.

    PubMed

    Min, Stephen K; Samaranayake, Chaminda P; Sastry, Sudhir K

    2011-05-26

    Direct measurements of reaction volume, so far, have been limited to atmospheric pressure. This study describes a method for in situ reaction volume measurements under pressure using a variable volume piezometer. Reaction volumes for protonic ionization of weak acid buffering agents (MES, citric acid, sulfanilic acid, and phosphoric acid) were measured in situ under pressure up to 400 MPa at 25 °C. The methodology involved initial separation of buffering agents within the piezometer using gelatin capsules. Under pressure, the volume of the reactants was measured at 25 °C, and the contents were heated to 40 °C to dissolve the gelatin and allow the reaction to occur, and cooled to 25 °C, where the volume of products was measured. Reaction volumes were used to calculate pH of the buffer solutions as a function of pressure. The results show that the measured reaction volumes as well as the calculated pH values generally quite agree with their respective theoretically predicted values up to 100 MPa. The results of this study highlight the need for a comprehensive theory to describe the pressure behavior of ionization reactions in realistic systems especially at higher pressures. PMID:21542618

  12. Humic Acid Complexation of Th, Hf and Zr in Ligand Competition Experiments: Metal Loading and Ph Effects

    NASA Technical Reports Server (NTRS)

    Stern, Jennifer C.; Foustoukos, Dionysis I.; Sonke, Jeroen E.; Salters, Vincent J. M.

    2014-01-01

    The mobility of metals in soils and subsurface aquifers is strongly affected by sorption and complexation with dissolved organic matter, oxyhydroxides, clay minerals, and inorganic ligands. Humic substances (HS) are organic macromolecules with functional groups that have a strong affinity for binding metals, such as actinides. Thorium, often studied as an analog for tetravalent actinides, has also been shown to strongly associate with dissolved and colloidal HS in natural waters. The effects of HS on the mobilization dynamics of actinides are of particular interest in risk assessment of nuclear waste repositories. Here, we present conditional equilibrium binding constants (Kc, MHA) of thorium, hafnium, and zirconium-humic acid complexes from ligand competition experiments using capillary electrophoresis coupled with ICP-MS (CE- ICP-MS). Equilibrium dialysis ligand exchange (EDLE) experiments using size exclusion via a 1000 Damembrane were also performed to validate the CE-ICP-MS analysis. Experiments were performed at pH 3.5-7 with solutions containing one tetravalent metal (Th, Hf, or Zr), Elliot soil humic acid (EHA) or Pahokee peat humic acid (PHA), and EDTA. CE-ICP-MS and EDLE experiments yielded nearly identical binding constants for the metal- humic acid complexes, indicating that both methods are appropriate for examining metal speciation at conditions lower than neutral pH. We find that tetravalent metals form strong complexes with humic acids, with Kc, MHA several orders of magnitude above REE-humic complexes. Experiments were conducted at a range of dissolved HA concentrations to examine the effect of [HA]/[Th] molar ratio on Kc, MHA. At low metal loading conditions (i.e. elevated [HA]/[Th] ratios) the ThHA binding constant reached values that were not affected by the relative abundance of humic acid and thorium. The importance of [HA]/[Th] molar ratios on constraining the equilibrium of MHA complexation is apparent when our estimated Kc, MHA values

  13. Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

    SciTech Connect

    Cingolani, Gino Andrews, Dewan; Casjens, Sherwood

    2006-05-01

    The crystallogenesis of bacteriophage P22 tail-fiber gp26 is described. To study possible pH-induced conformational changes in gp26 structure, native trimeric gp26 has been crystallized at acidic pH (4.6) and a chimera of gp26 fused to maltose-binding protein (MBP-gp26) has been crystallized at neutral and alkaline pH (7-10). Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26 forms an ∼220 Å extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell’s outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 Å resolution and belong to space group P2{sub 1}, with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high-throughput crystallization robot at the Hauptman–Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 Å resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress.

  14. MICROBIAL SULFATE REDUCTION AND METAL ATTENUATION IN PH 4 ACID MINE WATER

    EPA Science Inventory

    Sediments recovered from the flooded mine workings of the Penn Mine, a Cu-Zn mine abandoned since the early 1960s, were cultured for anaerobic bacteria over a range of pH (4 to 7.5). The molecular biology of sediments and cultures was studied to determine whether sulfate-reducing...

  15. Structure and oxidation state of hematite surfaces reacted with aqueous Fe(II) at acidic and neutral pH

    NASA Astrophysics Data System (ADS)

    Catalano, Jeffrey G.; Fenter, Paul; Park, Changyong; Zhang, Zhan; Rosso, Kevin M.

    2010-03-01

    Structural changes and surface oxidation state were examined following the reaction of hematite (0 0 1), (0 1 2), and (1 1 0) with aqueous Fe(II). X-ray reflectivity measurements indicated that Fe(II) induces changes in the structure of all three surfaces under both acidic (pH 3) and neutral (pH 7) conditions. The structural changes were generally independent of pH although the extent of surface transformation varied slightly between acidic and neutral conditions; no systematic trends with pH were observed. Induced changes on the (1 1 0) and (0 1 2) surfaces include the addition or removal of partial surface layers consistent with either growth or dissolution. In contrast, a <1 nm thick, discontinuous film formed on the (0 0 1) surface that appears to be epitaxial yet is not a perfect extension of the underlying hematite lattice, being either structurally defective, compositionally distinct, or nanoscale in size and highly relaxed. Resonant anomalous X-ray reflectivity measurements determined that the surface concentration of Fe(II) present after reaction at pH 7 was below the detection limit of approximately 0.5-1 μmol/m 2 on all surfaces. These observations are consistent with Fe(II) oxidative adsorption, whereby adsorbed Fe(II) is oxidized by structural Fe(III) in the hematite lattice, with the extent of this reaction controlled by surface structure at the atomic scale. The observed surface transformations at pH 3 show that Fe(II) oxidatively adsorbs on hematite surfaces at pH values where little net adsorption occurs, based on historical macroscopic Fe(II) adsorption behavior on fine-grained hematite powders. This suggests that Fe(II) plays a catalytic role, in which an electron from an adsorbed Fe(II) migrates to and reduces a lattice Fe(III) cation elsewhere, which subsequently desorbs in a scenario with zero net reduction and zero net adsorption. Given the general pH-independence and substantial mass transfer involved, this electron and atom exchange

  16. Simultaneous recordings of oesophageal acid exposure with conventional pH monitoring and a wireless system (Bravo)

    PubMed Central

    des Varannes, S Bruley; Mion, F; Ducrotté, P; Zerbib, F; Denis, P; Ponchon, T; Thibault, R; Galmiche, J P

    2005-01-01

    Objectives: Oesophageal pH monitoring is a useful test for the diagnosis of gastro-oesophageal reflux disease (GORD) but has some limitations related to the nasopharyngeal electrode. Recently, a telemetric catheter free system (CFS) (Bravo; Medtronic) was developed. The aim of this study was to determine the concordance of data between the conventional pH measurement system (CPHMS) and the CFS Bravo. Methods: Forty patients with symptoms suggestive of GORD underwent 24 hour oesophageal pH monitoring using the CPHMS with a nasopharyngeal electrode and the Bravo CFS simultaneously. The sensitive tips of both electrodes were positioned at the same level under fluoroscopy. In addition to automatic analysis, each reflux episode was checked visually and characterised. Results: There was a significant correlation (r = 0.87, p<0.0001) between the 24 hour oesophageal acid exposures recorded by the CPHMS and the CFS. Twenty four hour oesophageal acid exposure was significantly lower with the CFS than with the CPHMS (2.4 (0.4–8.7) v 3.6 (0.7–8.6); p< 0.0001). Consequently, with the CFS, the cut off level for the diagnosis of GORD, as calculated from the regression equation, was 2.9% (for the 4.2% cut off determined in controls with the CPHMS). After this adjustment, concordance of the diagnosis of GORD was 88% (kappa 0.760). Diagnosis of GORD was established in more patients with the CFS 48 hour results than with the 24 hour results. Conclusions: Despite strong correlations between oesophageal acid exposure recorded with the two devices, the Bravo CFS significantly under recorded acid exposure compared with the CPHMS. Provided some correcting factors are used, the Bravo CFS can improve the sensitivity of pHmetry for the diagnosis of GORD by allowing more prolonged recordings. PMID:15843417

  17. Effects of pH and fulvic acids concentration on the stability of fulvic acids--cerium (IV) oxide nanoparticle complexes.

    PubMed

    Oriekhova, Olena; Stoll, Serge

    2016-02-01

    The behavior of cerium (IV) oxide nanoparticles has been first investigated at different pH conditions. The point of zero charge was determined as well as the stability domains using dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. A baseline hydrodynamic diameter of 180 nm was obtained indicating that individual CeO2 nanoparticles are forming small aggregates. Then we analyzed the particle behavior at variable concentrations of fulvic acids for three different pH-electrostatic scenarios corresponding to positive, neutral and negative CeO2 surface charges. The presence of fulvic acids was found to play a key role on the CeO2 stability via the formation of electrostatic complexes. It was shown that a small amount of fulvic acids (2 mg L(-1)), representative of environmental fresh water concentrations, is sufficient to stabilize CeO2 nanoparticles (50 mg L(-1)). When electrostatic complexes are formed between negatively charged FAs and positively charged CeO2 NPs the stability of such complexes is obtained with time (up to 7 weeks) as well as in pH changing conditions. Based on zeta potential variations we also found that the fulvic acids are changing the CeO2 acid-base surface properties. Obtained results presented here constitute an important outcome in the domain of risk assessment, transformation and removal of engineered nanomaterials released into the environment. PMID:26347935

  18. Effects of pH on photochemical decomposition of perfluorooctanoic acid in different atmospheres by 185nm vacuum ultraviolet.

    PubMed

    Wang, Yuan; Zhang, Pengyi

    2014-11-01

    Perfluorooctanoic acid (PFOA), a persistent organic pollutant, receives increasing concerns due to its worldwide occurrence and resistance to most conventional treatment processes. The photochemical decomposition by 185nm vacuum ultraviolet (VUV) is one of the efficient methods for PFOA decomposition. The effects of pH on PFOA decomposition in nitrogen atmosphere or oxygen atmosphere were investigated. At its original pH (4.5) of PFOA aqueous solution, PFOA decomposed efficiently both in nitrogen and in oxygen atmosphere. However, when the pH increased to 12.0, PFOA decomposition was greatly inhibited in oxygen atmosphere, while it was greatly accelerated in nitrogen atmosphere with a very short half-life time (9min). Furthermore, fluorine atoms originally contained in PFOA molecules were almost completely transformed into fluoride ions. Two decomposition pathways have been proposed to explain the PFOA decomposition under different conditions. In acidic and neutral solutions, PFOA predominantly decomposes via the direct photolysis in both atmospheres; while in the alkaline solution and in the absence of oxygen, the decomposition of PFOA is mainly induced by hydrated electrons. PMID:25458674

  19. β2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH

    PubMed Central

    Goodchild, Sophia C.; Sheynis, Tania; Thompson, Rebecca; Tipping, Kevin W.; Xue, Wei-Feng; Ranson, Neil A.; Beales, Paul A.; Hewitt, Eric W.; Radford, Sheena E.

    2014-01-01

    Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of β2-microglobulin (β2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which β2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of β2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that β2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between β2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of β2m amyloid-associated osteoarticular tissue destruction in DRA. PMID:25100247

  20. In vivo measurements of changes in pH triggered by oxalic acid in leaf tissue of transgenic oilseed rape.

    PubMed

    Zou, Qiu-Ju; Liu, Sheng-Yi; Dong, Xu-Yan; Bi, Yan-Hua; Cao, Yuan-Cheng; Xu, Qiao; Zhao, Yuan-Di; Chen, Hong

    2007-01-01

    Oxalic acid (OA), a non-host-specific toxin secreted by Sclerotinia sclerotiorum during pathogenesis, has been demonstrated to be a major phytotoxic and pathogenic factor. Oxalate oxidase (OXO) is an enzyme associated with the detoxification of OA, and hence the introduction of an OXO gene into oilseed rape (Brassica napus L.) to break down OA may be an alternative way of increasing the resistance of the plant to Sclerotinia sclerotiorum. In order to investigate the activation of OXO in transgenic oilseed rape, a convenient and accessible method was used to monitor changes in pH in response to stress induced by OA. The pH sensor, a platinum microcylinder electrode modified using polyaniline film, exhibited a linear response within the pH range from 3 to 7, with a Nernst response slope of 70 mV/pH at room temperature. The linear correlation coefficient was 0.9979. Changes induced by OA in the pH values of leaf tissue of different oilseed rape species from Brassica napus L. were monitored in real time in vivo using this electrode. The results clearly showed that the transgenic oilseed rape was more resistant to OA than non-transgenic oilseed rape. PMID:17623369

  1. Assessment of Envi-Carb™ as a passive sampler binding phase for acid herbicides without pH adjustment.

    PubMed

    Seen, Andrew; Bizeau, Oceane; Sadler, Lachlan; Jordan, Timothy; Nichols, David

    2014-05-01

    The graphitised carbon solid phase extraction (SPE) sorbent Envi-Carb has been used to fabricate glass fibre filter- Envi-Carb "sandwich" disks for use as a passive sampler for acid herbicides. Passive sampler uptake of a suite of herbicides, including the phenoxyacetic acid herbicides 4-chloro-o-tolyloxyacetic acid (MCPA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 3,6-dichloro-2-methoxybenzoic acid (Dicamba), was achieved without pH adjustment, demonstrating for the first time a suitable binding phase for passive sampling of acid herbicides at neutral pH. Passive sampling experiments with Duck River (Tasmania, Australia) water spiked at 0.5 μg L(-1) herbicide concentration over a 7 d deployment period showed that sampling rates in Duck River water decreased for seven out of eight herbicides, and in the cases of 3,6-dichloro-2-pyridinecarboxylic acid (Clopyralid) and Dicamba no accumulation of the herbicides occurred in the Envi-Carb over the deployment period. Sampling rates for 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (Picloram), 2,4-D and MCPA decreased to approximately 30% of the sampling rates in ultrapure water, whilst sampling rates for 2-(4,6-dimethylpyrimidin-2-ylcarbamoylsulfamoyl) benzoic acid, methyl ester (Sulfometuron-methyl) and 3,5,6-Trichloro-2-pyridinyloxyacetic acid (Triclopyr) were approximately 60% of the ultrapure water sampling rate. For methyl N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-D-alaninate (Metalaxyl-M) there was little variation in sampling rate between passive sampling experiments in ultrapure water and Duck River water. SPE experiments undertaken with Envi-Carb disks using ultrapure water and filtered and unfiltered Duck River water showed that not only is adsorption onto particulate matter in Duck River water responsible for a reduction in herbicide sampling rate, but interactions of herbicides with dissolved or colloidal matter (matter able to pass through a 0.2 μm membrane filter) also reduces the herbicide sampling

  2. The effect of humic acid on the aggregation of titanium dioxide nanoparticles under different pH and ionic strengths.

    PubMed

    Zhu, Miao; Wang, Hongtao; Keller, Arturo A; Wang, Tao; Li, Fengting

    2014-07-15

    With the increasingly widespread use of titanium dioxide nanoparticles (TiO2 NPs), the particles' environmental impacts have attracted concern, making it necessary to understand the fate and transport of TiO2 NPs in aqueous media. In this study, we investigated TiO2 NP aggregation caused by the effects of humic acid (HA), ionic strength (IS) and different pH using dynamic light scattering (DLS) to monitor the size distribution of the TiO2 NPs continuously. It was determined that HA can influence the stability of TiO2 NPs through charge neutralization, steric hindrance and bridging effects. In the absence of IS, aggregation was promoted by adding HA only when the pH (pH=4) is less than the point of zero charge for the TiO2 NPs (pHPZC≈6) because HA reduces the zeta potential of the TiO2 NPs via charge neutralization. At pH=4 and when the concentration of HA is 94.5 μg/L, the zeta potential of TiO2 NPs is close to zero, and they reach an aggregation maximum. A higher concentration of HA results in more negatively charged TiO2 NP surfaces, which hinder their aggregation. When the pH is 5.8, HA enhances the negative zeta potential of the TiO2 NPs and increases their stability via electrostatic repulsion and steric hindrance. When the pH (pH=8) is greater than pHpzc, the zeta potential of the TiO2 NPs is high (~40 mV), and it barely changes with increasing HA concentration. Thus, the TiO2 NPs are notably stable, and their size does not grow at pH8. The increase in the critical coagulation concentration (CCC) of TiO2 NPs indicated that there is steric hindrance after the addition of HA. HA can enhance the coagulation of TiO2 NPs, primarily due to bridging effect. These findings are useful in understanding the size change of TiO2 NPs, as well as the removal of TiO2 NPs and HA from aqueous media. PMID:24793841

  3. Inhibition of DNA adduct formation of PhIP in female F344 rats by dietary conjugated linoleic acid.

    PubMed

    Josyula, S; He, Y H; Ruch, R J; Schut, H A

    1998-01-01

    The dietary mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary carcinogen in the female Fischer (F344) rat and a colon carcinogen in the male F344 rat. To exert its carcinogenicity, it is believed that PhIP needs to form adducts with DNA, a process requiring N-hydroxylation of PhIP by cytochromes P-450 1A1 and/or 1A2 (CYP 1A1 and/or 1A2), as well as further esterification of the hydroxylamine thus formed. Dietary conjugated linoleic acid (CLA) inhibits chemical carcinogenesis in various experimental models. We have examined the effect of dietary CLA on PhIP-DNA adduct formation in female F344 rats. Four-week-old animals were maintained on AIN-76A diet without or with CLA (1%, 0.5%, and 0.1% wt/wt) for 57 days. PhIP was added to the diets (0.04% wt/wt) from Days 14-42. Animals were killed (4/group) on Days 43, 50, and 57. DNA isolated from liver, mammary epithelial cells (MEC), colon, and white blood cells (WBC) was analyzed for PhIP-DNA adducts by 32P-postlabeling assays. On Day 43, CLA inhibited adduct formation in the liver (up to 58%) in a dose-dependent manner. CLA also inhibited hepatic adduct levels (29-39%) on Day 50 (at 1.0% and 0.5% CLA) and on Day 57 (53% at 0.5% CLA). CLA significantly reduced adduct levels in the WBC on Day 50 (63-70%). Adducts in MEC and the colon were not affected by dietary CLA. On Day 57, adduct levels in MEC, liver, colon, and WBC were 0-30.3%, 8.6-41.7%, 21.5-50.7%, and 7.5-11.8%, respectively, of those on Day 43. Northern blot analysis of liver RNA showed that dietary CLA did not affect steady-state levels of CYP 1A1 or 1A2 mRNA. It is concluded that dietary CLA inhibits PhIP-DNA adduct formation in liver and WBC but that those in MEC and the colon are unaffected when a low-level dietary regimen of carcinogen and inhibitor was used. In inhibiting PhIP-DNA adduct formation, CLA does not appear to act by inhibiting CYP 1A1 or 1A2 expression. PMID:10050262

  4. Salinity and pH affect Na+-montmorillonite dissolution and amino acid adsorption: a prebiotic chemistry study

    NASA Astrophysics Data System (ADS)

    Farias, Ana Paula S. F.; Tadayozzi, Yasmin S.; Carneiro, Cristine E. A.; Zaia, Dimas A. M.

    2014-06-01

    The adsorption of amino acids onto minerals in prebiotic seas may have played an important role for their protection against hydrolysis and formation of polymers. In this study, we show that the adsorption of the prebiotic amino acids, glycine (Gly), α-alanine (α-Ala) and β-alanine (β-Ala), onto Na+-montmorillonite was dependent on salinity and pH. Specifically, adsorption decreased from 58.3-88.8 to 0-48.9% when salinity was increased from 10 to 100-150% of modern seawater. This result suggests reduced amino acid adsorption onto minerals in prebiotic seas, which may have been even more saline than the tested conditions. Amino acids also formed complexes with metals in seawater, affecting metal adsorption onto Na+-montmorillonite, and amino acid adsorption was enhanced when added before Na+-montmorillonite was exposed to high saline solutions. Also, the dissolution of Na+-montmorillonite was reduced in the presence of amino acids, with β-Ala being the most effective. Thus, prebiotic chemistry experiments should also consider the integrity of minerals in addition to their adsorption capacity.

  5. Intracellular acidification is required for full activation of the sweet taste receptor by miraculin.

    PubMed

    Sanematsu, Keisuke; Kitagawa, Masayuki; Yoshida, Ryusuke; Nirasawa, Satoru; Shigemura, Noriatsu; Ninomiya, Yuzo

    2016-01-01

    Acidification of the glycoprotein, miraculin (MCL), induces sweet taste in humans, but not in mice. The sweet taste induced by MCL is more intense when acidification occurs with weak acids as opposed to strong acids. MCL interacts with the human sweet receptor subunit hTAS1R2, but the mechanisms by which the acidification of MCL activates the sweet taste receptor remain largely unexplored. The work reported here speaks directly to this activation by utilizing a sweet receptor TAS1R2 + TAS1R3 assay. In accordance with previous data, MCL-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of MCL was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of MCL was proportional to intracellular pH regardless of types of acids. These results suggest that intracellular acidity is required for full activation of the sweet taste receptor by MCL. PMID:26960429

  6. Intracellular acidification is required for full activation of the sweet taste receptor by miraculin

    PubMed Central

    Sanematsu, Keisuke; Kitagawa, Masayuki; Yoshida, Ryusuke; Nirasawa, Satoru; Shigemura, Noriatsu; Ninomiya, Yuzo

    2016-01-01

    Acidification of the glycoprotein, miraculin (MCL), induces sweet taste in humans, but not in mice. The sweet taste induced by MCL is more intense when acidification occurs with weak acids as opposed to strong acids. MCL interacts with the human sweet receptor subunit hTAS1R2, but the mechanisms by which the acidification of MCL activates the sweet taste receptor remain largely unexplored. The work reported here speaks directly to this activation by utilizing a sweet receptor TAS1R2 + TAS1R3 assay. In accordance with previous data, MCL-applied cells displayed a pH dependence with citric acid (weak acid) being right shifted to that with hydrochloric acid (strong acid). When histidine residues in both the intracellular and extracellular region of hTAS1R2 were exchanged for alanine, taste-modifying effect of MCL was reduced or abolished. Stronger intracellular acidification of HEK293 cells was induced by citric acid than by HCl and taste-modifying effect of MCL was proportional to intracellular pH regardless of types of acids. These results suggest that intracellular acidity is required for full activation of the sweet taste receptor by MCL. PMID:26960429

  7. Interactions of Zn(II) Ions with Humic Acids Isolated from Various Type of Soils. Effect of pH, Zn Concentrations and Humic Acids Chemical Properties

    PubMed Central

    Boguta, Patrycja; Sokołowska, Zofia

    2016-01-01

    The main aim of this study was the analysis of the interaction between humic acids (HAs) from different soils and Zn(II) ions at wide concentration ranges and at two different pHs, 5 and 7, by using fluorescence and FTIR spectroscopy, as well as potentiometric measurements. The presence of a few areas of HAs structures responsible for Zn(II) complexing was revealed. Complexation at α-sites (low humified structures of low-molecular weight and aromatic polycondensation) and β-sites (weakly humified structures) was stronger at pH 7 than 5. This trend was not observed for γ-sites (structures with linearly-condensed aromatic rings, unsaturated bonds and large molecular weight). The amount of metal complexed at pH5 and 7 by α and γ-structures increased with a decrease in humification and aromaticity of HAs, contrary to β-areas where complexation increased with increasing content of carboxylic groups. The stability of complexes was higher at pH 7 and was the highest for γ-structures. At pH 5, stability decreased with C/N increase for α-areas and -COOH content increase for β-sites; stability increased with humification decrease for γ-structures. The stability of complexes at α and β-areas at pH 7 decreased with a drop in HAs humification. FTIR spectra at pH 5 revealed that the most-humified HAs tended to cause bidentate bridging coordination, while in the case of the least-humified HAs, Zn caused bidentate bridging coordination at low Zn additions and bidentate chelation at the highest Zn concentrations. Low Zn doses at pH 7 caused formation of unidentate complexes while higher Zn doses caused bidentate bridging. Such processes were noticed for HAs characterized by high oxidation degree and high oxygen functional group content; where these were low, HAs displayed bidentate bridging or even bidentate chelation. To summarize, the above studies have showed significant impact of Zn concentration, pH and some properties of HAs on complexation reactions of humic

  8. Photonic crystal fiber interferometric pH sensor based on polyvinyl alcohol/polyacrylic acid hydrogel coating.

    PubMed

    Hu, Pengbing; Dong, Xinyong; Wong, Wei Chang; Chen, Li Han; Ni, Kai; Chan, Chi Chiu

    2015-04-01

    We present a simple photonic crystal fiber interferometer (PCFI) that operates in reflection mode for pH measurement. The sensor is made by coating polyvinyl alcohol/polyacrylic acid (PVA/PAA) hydrogel onto the surface of the PCFI, constructed by splicing a stub of PCF at the distal end of a single-mode fiber with its free end airhole collapsed. The experimental results demonstrate a high average sensitivity of 0.9 nm/pH unit for the 11 wt.% PVA/PAA coated sensor in the pH range from 2.5 to 6.5. The sensor also displays high repeatability and stability and low cross-sensitivity to temperature. Fast, reversible rise and fall times of 12 s and 18 s, respectively, are achieved for the sensor time response. PMID:25967171

  9. Low pH, aluminum and phosphorus coordinately regulate malate exudation through GmALMT1 to improve soybean adaptation to acid soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low pH, aluminum (Al) toxicity and low phosphorus (P) often coexist in acid soils where crops need to cope with these multiple limiting factors. In this study we found that P addition to acid soils alleviates Al toxicity and enhanced soybean adaptation to acid soils, especially for the P-efficient g...

  10. Polymerization of acrylamide at acid pH using uranyl nitrate

    SciTech Connect

    Deshpande, V.V.; Bodhe, A.M.; Pawar, H.S.; Vartak, H.G.

    1986-03-01

    A new photopolymerizing reagent, uranyl nitrate, is used for the polymerization of acrylamide gels at low pH. The amount of uranyl nitrate (0.2 mg/ml) required for the polymerization of gels at pH 3.0 is considerably less than that of persulfate (7 mg/ml). Use of this reagent obviates the need for the removal of excess of persulfate by preelectrophoresis. The electrophoretic separation of basic proteins in uranium-polymerized gels showed faster movement and better resolution of proteins and proved the gels to be versatile, uniform, and reproducible. Electrophoresis of trypsin in these gels does not affect the enzymatic activity. The catalyst can also be used for the polymerization of gels containing 3 M urea.

  11. Aluminum tolerance of two wheat cultivars (Brevor and Atlas66) in relation to their rhizosphere pH and organic acids exuded from roots.

    PubMed

    Wang, Ping; Bi, Shuping; Ma, Liping; Han, Weiying

    2006-12-27

    Phytotoxicity of aluminum (Al) has become a serious problem in inhibiting plant growth on acid soils. Under Al stress, the changes of rhizosphere pH, root elongation, absorption of Al by wheat roots, organic acids exuded from roots, and some main factors related to Al-tolerant mechanisms have been studied using hydroponics, fluorescence spectrophotometry, and high performance liquid chromatography (HPLC). Two wheat cultivars, Brevor and Atlas66, differing in Al tolerance are chosen in the study. Accordingly, the rhizosphere pH has a positive effect on Al tolerance. Atlas66 (Al-tolerant) has higher capability to maintain high rhizosphere pH than Brevor (Al-sensitive) does. High pH can reduce Al3+ activity and toxicity, and increase the efficiency of exuding organic acids from the roots. More inhibition of root elongation has been found in Brevor because of the exposure of roots to Al3+ solution at low pH. Brevor accumulate more Al in roots than Atlas66 even at higher pH. Al-induced exudation of malic and citric acids has been found in Atlas66 roots, while no Al-induced organic acids have been found in Brevor. These results indicate that the Al-induced secretion of organic acids from Atlas66 roots has a positive correlation with Al tolerance. Comprehensive treatment of Al3+ and H+ indicates that wheat is adversely influenced by excess Al3+, rather than low pH. PMID:17177538

  12. Enhancing the intestinal absorption of poorly water-soluble weak-acidic compound by controlling local pH.

    PubMed

    Iwanaga, Kazunori; Kato, Shino; Miyazaki, Makoto; Kakemi, Masawo

    2013-12-01

    Recently, the number of poorly water-soluble drug candidates has increased and has hindered the rapid improvement of new drugs with low intestinal absorption; however, the intestinal absorption of pH-dependent poorly water-soluble compounds is expected to be markedly improved by changing the pH in the vicinity of the absorption site. The aim of this study is to clarify the effect of local pH change in the intestinal tract by magnesium oxide on the intestinal absorption of hydrochlorothiazide, a model poorly water-soluble weak-acid compound. The application of hydrochlorothiazide granule containing magnesium oxide to the rat intestinal loop increased the pH in the vicinity of the dosing site to more than 8.5 for 90 min without any mucosal damage. As a result, absorption of hydrochlorothiazide increased by the addition of magnesium oxide to the granule. Intraintestinal administration of a suspension prepared from hydrochlorothiazide granules with magnesium oxide increased the intestinal absorption and the AUC value was 3-fold higher than that without magnesium oxide. To further increase the intestinal absorption of hydrochlorothiazide, we prepared granules containing magnesium oxide and chitosan as a mucoadhesive and tight junction opening material. Chitosan showed a marked increase of intestinal absorption, and the AUC value after the administration of suspensions of chitosan granules was more than 5-fold higher than that of granules containing hydrochlorothiazide alone, respectively. In summary, it has been clarified that the intestinal absorption of weak-acidic poorly water-soluble compound can be enhanced by increasing local pH, mucoadhesion and opening tight junction. PMID:22443480

  13. Systematics and species-specific response to pH of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) from acid mining lakes.

    PubMed

    Weisse, Thomas; Moser, Michael; Scheffel, Ulrike; Stadler, Peter; Berendonk, Thomas; Weithoff, Guntram; Berger, Helmut

    2013-05-01

    We investigated the morphology, phylogeny of the 18S rDNA, and pH response of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) isolated from two chemically similar acid mining lakes (pH~2.6) located at Langau, Austria, and in Lusatia, Germany. Oxytricha acidotolerans sp. nov. from Langau has 18 frontal-ventral-transverse cirri but a very indistinct kinety 3 fragmentation so that the assignment to Oxytricha is uncertain. The somewhat smaller species from Lusatia has a highly variable cirral pattern and the dorsal kineties arranged in the Urosomoida pattern and is, therefore, preliminary designated as Urosomoida sp. The pH response was measured as ciliate growth rates in laboratory experiments at pH ranging from 2.5 to 7.0. Our hypothesis was that the shape of the pH reaction norm would not differ between these closely related (3% difference in their SSU rDNA) species. Results revealed a broad pH niche for O. acidotolerans, with growth rates peaking at moderately acidic conditions (pH 5.2). Cyst formation was positively and linearly related to pH. Urosomoida sp. was more sensitive to pH and did not survive at circumneutral pH. Accordingly, we reject our hypothesis that similar habitats would harbour ciliate species with virtually identical pH reaction norm. PMID:23021638

  14. Poly-dopamine-beta-cyclodextrin: A novel nanobiopolymer towards sensing of some amino acids at physiological pH.

    PubMed

    Hasanzadeh, Mohammad; Sadeghi, Sattar; Bageri, Leyla; Mokhtarzadeh, Ahad; Karimzadeh, Ayub; Shadjou, Nasrin; Mahboob, Soltanali

    2016-12-01

    A novel nanobiopolymer film was electrodeposited on the surface of glassy carbon through cyclic voltammetry from dopamine, β-cyclodextrin, and phosphate buffer solution in physiological pH (7.40). The electrochemical behavior of polydopamine-Beta-cyclodextrin modified glassy carbon electrode was investigated for electro-oxidation and determination of some amino acids (l-Cysteine, l-Tyrosine, l-Glycine, and l-Phenylalanine). The modified electrode was applied for selected amino acid detection at physiological pH using cyclic voltammetry, differential pulse voltammetry and chronoamperometry, chronocoulometery. The linear concentration range of the proposed sensor for the l-Glycine, l-Cysteine, l-Tyrosine, and l-Phenylalanine were 0.2-70, 0.06-0.2, 0.01-0.1, and 0.2-10μM, while low limit of quantifications were 0.2, 0.06, 0.01, and 0.2μM, respectively. The modified electrode shows many advantages as an amino acid sensor such as simple preparation method without using any specific electron transfer mediator or specific reagent, good sensitivity, short response time, and long term stability. PMID:27612722

  15. AN HPLC METHOD WITH UV DETECTION, PH CONTROL, AND REDUCTIVE ASCORBIC ACID FOR CYANURIC ACID ANALYSIS IN WATER

    EPA Science Inventory

    Every year over 250 million pounds of cyanuric acid (CA) and chloroisocyanurates are produced industrially. These compounds are standard ingredients in formulations for household bleaches, industrial cleansers, dishwasher compounds, general sanitizers, and chlorine stabilizers. ...

  16. AN HPLC METHOD WITH UVDETECTION, PH CONTROL, AND REDUCTIVE ASCORBIC ACID FOR CYANURIC ACID ANALYSIS IN WATER

    EPA Science Inventory

    Every year over 250 million pounds of cyanuric acid (CA) and chlorinated isocyanurates are produced industrially. These compounds are standard ingredients in formulations for household bleaches, industrial cleansers, dishwasher compounds, general sanitizers, and chlorine stabiliz...

  17. Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low pH

    PubMed Central

    Cotter, Paul D.; Hill, Colin

    2003-01-01

    Gram-positive bacteria possess a myriad of acid resistance systems that can help them to overcome the challenge posed by different acidic environments. In this review the most common mechanisms are described: i.e., the use of proton pumps, the protection or repair of macromolecules, cell membrane changes, production of alkali, induction of pathways by transcriptional regulators, alteration of metabolism, and the role of cell density and cell signaling. We also discuss the reponses of Listeria monocytogenes, Rhodococcus, Mycobacterium, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, oral streptococci, and lactic acid bacteria to acidic environments and outline ways in which this knowledge has been or may be used to either aid or prevent bacterial survival in low-pH environments. PMID:12966143

  18. Two distinct etiologies of gastric cardia adenocarcinoma: interactions among pH, Helicobacter pylori, and bile acids

    PubMed Central

    Mukaisho, Ken-ichi; Nakayama, Takahisa; Hagiwara, Tadashi; Hattori, Takanori; Sugihara, Hiroyuki

    2015-01-01

    Gastric cancer can be classified as cardia and non-cardia subtypes according to the anatomic site. Although the gastric cancer incidence has decreased steadily in several countries over the past 50 years, the incidence of cardia cancers and esophageal adenocarcinoma (EAC) continue to increase. The etiological factors involved in the development of both cardia cancers and EACs are associated with high animal fat intake, which causes severe obesity. Central obesity plays roles in cardiac-type mucosa lengthening and partial hiatus hernia development. There are two distinct etiologies of cardia cancer subtypes: one associated with gastroesophageal reflux (GER), which predominantly occurs in patients without Helicobacter pylori (H. pylori) infection and resembles EAC, and the other associated with H. pylori atrophic gastritis, which resembles non-cardia cancer. The former can be developed in the environment of high volume duodenal content reflux, including bile acids and a higher acid production in H. pylori–negative patients. N-nitroso compounds, which are generated from the refluxate that includes a large volume of bile acids and are stabilized in the stomach (which has high levels of gastric acid), play a pivotal role in this carcinogenesis. The latter can be associated with the changing colonization of H. pylori from the distal to the proximal stomach with atrophic gastritis because a high concentration of soluble bile acids in an environment of low acid production is likely to act as a bactericide or chemorepellent for H. pylori in the distal stomach. The manuscript introduces new insights in causative factors of adenocarcinoma of the cardia about the role of bile acids in gastro-esophageal refluxate based upon robust evidences supporting interactions among pH, H. pylori, and bile acids. PMID:26029176

  19. Experimental evaluation of the contribution of acidic pH and Fe concentration to the structure, function and tolerance to metals (Cu and Zn) exposure in fluvial biofilms.

    PubMed

    Luís, Ana Teresa; Bonet, Berta; Corcoll, Natàlia; Almeida, Salomé F P; da Silva, Eduardo Ferreira; Figueira, Etelvina; Guasch, Helena

    2014-09-01

    An indoor channel system was colonised with fluvial biofilms to study the chronic effects of high Fe and SO4(2-) concentrations and acidic pH, the water chemistry in the surrounding streams of Aljustrel mining area (Alentejo, Portugal), and their contribution to community (in)tolerance to metal toxicity by short-term experiments with Cu and Zn. Biofilms were subjected to four different treatments during 8 weeks: high Fe and SO4(2-) concentrations (1 mg Fe l(-1)+ 700 mg SO4(2-) l(-1)) and acidic pH, high Fe and SO4(2-) at alkaline pH; lower Fe and SO4(2-) at acidic pH: and lower Fe and SO4(2-) concentrations at alkaline pH as negative control. During chronic exposure, acidic pH affected growth negatively, based on low values of algal biomass and the autotrophic index, high values of the antioxidant enzyme activities and low diversity diatom communities, dominated by acidophilic species (Pinnularia aljustrelica) in acidic treatments, being the effects more marked with high Fe and SO4(2-). Co-tolerance to metals (Cu and Zn) was also shown in biofilms from the acidic treatments, contrasting with the higher sensitivity observed in the alkaline treatments. We can conclude that the Aljustrel mining area acidic environment limits algal growth and exerts a strong selection pressure on the community composition which is in turn, more tolerant to metal exposure. PMID:25011920

  20. Effect of salt and acidic pH on the stability of virulence plasmid (pYV) in Yersinia enterocolitica and expression of virulence-associated characteristics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The stability of the Yersinia enterocolitica virulence plasmid (pYV) under different NaCl concentrations and under acidic pH conditions was investigated. Exposure of five strains representing five serotypes of pYV-bearing virulent Y. enterocolitica to 0.5, 2 and 5% NaCl and under conditions of pH 4...

  1. Influence on Levels of Information as Presented by Different Technologies on Students' Understanding of Acid, Base, and pH Concepts.

    ERIC Educational Resources Information Center

    Nakhleh, Mary B.; Krajcik, Joseph S.

    1994-01-01

    Involves secondary students in a study designed to allow investigation into how different levels of information presented by various technologies (chemical indicators, pH meters, and microcomputer-based laboratories-MBLs) affected students' understanding of acid, base, and pH concepts. Results showed that students using MBLs exhibited a greater…

  2. The effects of temperature, pH and redox state on the stability of glutamic acid in hydrothermal fluids

    NASA Astrophysics Data System (ADS)

    Lee, Namhey; Foustoukos, Dionysis I.; Sverjensky, Dimitri A.; Cody, George D.; Hazen, Robert M.

    2014-06-01

    Natural hydrothermal vent environments cover a wide range of physicochemical conditions involving temperature, pH and redox state. The stability of simple biomolecules such as amino acids in such environments is of interest in various fields of study from the origin of life to the metabolism of microbes at the present day. Numerous previous experimental studies have suggested that amino acids are unstable under hydrothermal conditions and decompose rapidly. However, previous studies have not effectively controlled the redox state of the hydrothermal fluids. Here we studied the stability of glutamate with and without reducing hydrothermal conditions imposed by 13 mM aqueous H2 at temperatures of 150, 200 and 250 °C and initial (25 °C) pH values of 6 and 10 in a flow-through hydrothermal reactor with reaction times from 3 to 36 min. We combined the experimental measurements with theoretical calculations to model the in situ aqueous speciation and pH values. As previously observed under hydrothermal conditions, the main reaction involves glutamate cyclizing to pyroglutamate through a simple dehydration reaction. However, the amounts of decomposition products of the glutamate detected, including succinate, formate, carbon dioxide and ammonia depend on the temperature, the pH and particularly the redox state of the fluid. In the absence of dissolved H2, glutamate decomposes in the sequence glutamate, glutaconate, α-hydroxyglutarate, ketoglutarate, formate and succinate, and ultimately to CO2 and micromolar quantities of H2(aq). Model speciation calculations indicate the CO2, formate and H2(aq) are not in metastable thermodynamic equilibrium. However, with 13 mM H2(aq) concentrations, the amounts of decomposition products are suppressed at all temperatures and pH values investigated. The small amounts of CO2 and formate present are calculated to be in metastable equilibrium with the H2. It is further proposed that there is a metastable equilibrium between glutamate

  3. The role of carbon dioxide (and intracellular pH) in the pathomechanism of several mental disorders. Are the diseases of civilization caused by learnt behaviour, not the stress itself?

    PubMed

    Sikter, András; Faludi, Gábor; Rihmer, Zoltán

    2009-09-01

    The role of carbon dioxide (CO2) is underestimated in the pathomechanism of neuropsychiatric disorders, though it is an important link between psyche and corpus. The actual spiritual status also influences respiration (we start breathing rarely, frequently, irregularly, etc.) causing pH alteration in the organism; on the other hand the actual cytosolic pH of neurons is one of the main modifiers of Ca2+-conductance, hence breathing directly, quickly, and effectively influences the second messenger system through Ca2+-currents. (Decreasing pCO2 turns pH into alkalic direction, augments psychic arousal, while increasing pCO2 turns pH acidic, diminishes arousal.) One of the most important homeostatic function is to maintain or restore the permanence of H+-concentration, hence the alteration of CO2 level starts cascades of contraregulation. However it can be proved that there is no perfect compensation, therefore compensational mechanisms may generate psychosomatic disorders causing secondary alterations in the "milieu interieur". Authors discuss the special physico-chemical features of CO2, the laws of interweaving alterations of pCO2 and catecholamine levels (their feedback mechanism), the role of acute and chronic hypocapnia in several hyperarousal disorders (delirium, panic disorder, hyperventilation syndrome, generalized anxiety disorder, bipolar disorder), the role of "locus minoris resistentiae" in the pathomechanism of psychosomatic disorders. It is supposed that the diseases of civilization are caused not by the stress itself but the lack of human instinctive reaction to it, and this would cause long-lasting CO2 alteration. Increased brain-pCO2, acidic cytosol pH and/or increased basal cytosolic Ca2+ level diminish inward Ca2+-current into cytosol, decrease arousal--they may cause dysthymia or depression. This state usually co-exists with ATP-deficiency and decreased cytosolic Mg2+ content. This energetical- and ion-constellation is also typical of ageing

  4. Peptide-Modulated Activity Enhancement of Acidic Protease Cathepsin E at Neutral pH

    PubMed Central

    Komatsu, Masayuki; Biyani, Madhu; Ghimire Gautam, Sunita; Nishigaki, Koichi

    2012-01-01

    Enzymes are regulated by their activation and inhibition. Enzyme activators can often be effective tools for scientific and medical purposes, although they are more difficult to obtain than inhibitors. Here, using the paired peptide method, we report on protease-cathepsin-E-activating peptides that are obtained at neutral pH. These selected peptides also underwent molecular evolution, after which their cathepsin E activation capability improved. Thus, the activators we obtained could enhance cathepsin-E-induced cancer cell apoptosis, which indicated their potential as cancer drug precursors. PMID:23365585

  5. Time-dependent activity of Na+/H+ exchanger isoform 1 and homeostasis of intracellular pH in astrocytes exposed to CoCl2 treatment.

    PubMed

    Wang, Peng; Li, Ling; Zhang, Zhenxiang; Kan, Quancheng; Gao, Feng; Chen, Suyan

    2016-05-01

    Hypoxia causes injury to the central nervous system during stroke and has significant effects on pH homeostasis. Na+/H+ exchanger isoform 1 (NHE1) is important in the mechanisms of hypoxia and intracellular pH (pHi) homeostasis. As a well-established hypoxia-mimetic agent, CoCl2 stabilizes and increases the expression of hypoxia inducible factor‑1α (HIF-1α), which regulates several genes involved in pH balance, including NHE1. However, it is not fully understood whether NHE1 is activated in astrocytes under CoCl2 treatment. In the current study, pHi and NHE activity were analyzed using the pHi‑sensitive dye BCECF‑AM. Using cariporide (an NHE1‑specific inhibitor) and EIPA (an NHE nonspecific inhibitor), the current study demonstrated that it was NHE1, not the other NHE isoforms, that was important in regulating pHi homeostasis in astrocytes during CoCl2 treatment. Additionally, the present study observed that, during the early period of CoCl2 treatment (the first 2 h), NHE1 activity and pHi dropped immediately, and NHE1 mRNA expression was reduced compared with control levels, whereas expression levels of the NHE1 protein had not yet changed. In the later period of CoCl2 treatment, NHE1 activity and pHi significantly increased compared with the control levels, as did the mRNA and protein expression levels of NHE1. Furthermore, the cell viability and injury of astrocytes was not changed during the initial 8 h of CoCl2 treatment; their deterioration was associated with the higher levels of pHi and NHE1 activity. The current study concluded that NHE1 activity and pHi homeostasis are regulated by CoCl2 treatment in a time-dependent manner in astrocytes, and may be responsible for the changes in cell viability and injury observed under hypoxia-mimetic conditions induced by CoCl2 treatment. PMID:27035646

  6. Systematics and species-specific response to pH of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) from acid mining lakes

    PubMed Central

    Weisse, Thomas; Moser, Michael; Scheffel, Ulrike; Stadler, Peter; Berendonk, Thomas; Weithoff, Guntram; Berger, Helmut

    2013-01-01

    We investigated the morphology, phylogeny of the 18S rDNA, and pH response of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) isolated from two chemically similar acid mining lakes (pH ∼ 2.6) located at Langau, Austria, and in Lusatia, Germany. Oxytricha acidotolerans sp. nov. from Langau has 18 frontal-ventral-transverse cirri but a very indistinct kinety 3 fragmentation so that the assignment to Oxytricha is uncertain. The somewhat smaller species from Lusatia has a highly variable cirral pattern and the dorsal kineties arranged in the Urosomoida pattern and is, therefore, preliminary designated as Urosomoida sp. The pH response was measured as ciliate growth rates in laboratory experiments at pH ranging from 2.5 to 7.0. Our hypothesis was that the shape of the pH reaction norm would not differ between these closely related (3% difference in their SSU rDNA) species. Results revealed a broad pH niche for O. acidotolerans, with growth rates peaking at moderately acidic conditions (pH 5.2). Cyst formation was positively and linearly related to pH. Urosomoida sp. was more sensitive to pH and did not survive at circumneutral pH. Accordingly, we reject our hypothesis that similar habitats would harbour ciliate species with virtually identical pH reaction norm. PMID:23021638

  7. Silica precipitation in acidic solutions: mechanism, pH effect, and salt effect.

    PubMed

    Gorrepati, Elizabeth A; Wongthahan, Pattanapong; Raha, Sasanka; Fogler, H Scott

    2010-07-01

    This study is the first to show that silica precipitation under very acidic conditions ([HCl] = 2-8 M) proceeds through two distinct steps. First, the monomeric form of silica is quickly depleted from solution as it polymerizes to form primary particles approximately 5 nm in diameter. Second, the primary particles formed then flocculate. A modified Smoluchowski equation that incorporates a geometric population balance accurately describes the exponential growth of silica flocs. Variation of the HCl concentration between 2 and 8 M further showed that polymerization to form primary particles and subsequent particle flocculation become exponentially faster with increasing acid concentration. The effect of salt was also studied by adding 1 M chloride salts to the solutions; it was found that salts accelerated both particle formation and growth rates in the order: AlCl(3) > CaCl(2) > MgCl(2) > NaCl > CsCl > no salt. It was also found that ionic strength, over cation identity, determines silica polymerization and particle flocculation rates. This research reveals that precipitation of silica products from acid dissolution of minerals can be studied apart from the mineral dissolution process. Thus, silica product precipitation from mineral acidization follows a two-step process--formation of 5 nm primary particles followed by particle flocculation--which becomes exponentially faster with increasing HCl concentration and with salts accelerating the process in the above order. This result has implications for any study of acid dissolution of aluminosilicate or silicate material. In particular, the findings are applicable to the process of acidizing oil-containing rock formations, a common practice of the petroleum industry where silica dissolution products encounter a low-pH, salty environment within the oil well. PMID:20536253

  8. Nickel toxicity to microbes: effect of pH and implications for acid rain

    SciTech Connect

    Babich, H.; Stotzky, G.

    1982-12-01

    A broad spectrum of microorganisms, including eubacteria (nonmarine and marine), actinomycetes, yeasts, and filamentous fungi, were evaluated for their sensitivities to nickel. Wide extremes in sensitivity to Ni were noted among the filamentous fungi, whereas the range of tolerance to Ni of the yeasts, eubacteria, and actinomycetes was narrower. With all microorganisms, the toxicity of Ni has not been defined, although the formation of hydroxylated Ni species with differing toxicities was not involved. The enhanced toxicity of Ni at acidic levels may have implications for the toxicity of Ni in environments stressed by acid precipitation.

  9. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae)

    PubMed Central

    Shane, Michael W.; Stigter, Kyla; Fedosejevs, Eric T.; Plaxton, William C.

    2014-01-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native ‘extremophile’ plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors’ knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  10. Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae).

    PubMed

    Shane, Michael W; Stigter, Kyla; Fedosejevs, Eric T; Plaxton, William C

    2014-11-01

    Despite its agronomic importance, the metabolic networks mediating phosphorus (P) remobilization during plant senescence are poorly understood. Highly efficient P remobilization (~85%) from senescing leaves and proteoid roots of harsh hakea (Hakea prostrata), a native 'extremophile' plant of south-western Australia, was linked with striking up-regulation of cell wall-localized and intracellular acid phosphatase (APase) and RNase activities. Non-denaturing PAGE followed by in-gel APase activity staining revealed senescence-inducible 120kDa and 60kDa intracellular APase isoforms, whereas only the 120kDa isoform was detected in corresponding cell wall fractions. Kinetic and immunological properties of the 120kDa and 60kDa APases partially purified from senescing leaves indicated that they are purple acid phosphatases (PAPs). Results obtained with cell wall-targeted hydrolases of harsh hakea were corroborated using Arabidopsis thaliana in which an ~200% increase in cell wall APase activity during leaf senescence was paralleled by accumulation of immunoreactive 55kDa AtPAP26 polypeptides. Senescing leaves of an atpap26 T-DNA insertion mutant displayed a >90% decrease in cell wall APase activity. Previous research established that senescing leaves of atpap26 plants exhibited a similar reduction in intracellular (vacuolar) APase activity, while displaying markedly impaired P remobilization efficiency and delayed senescence. It is hypothesized that up-regulation and dual targeting of PAPs and RNases to the cell wall and vacuolar compartments make a crucial contribution to highly efficient P remobilization that dominates the P metabolism of senescing tissues of harsh hakea and Arabidopsis. To the best of the authors' knowledge, the apparent contribution of cell wall-targeted hydrolases to remobilizing key macronutrients such as P during senescence has not been previously suggested. PMID:25170100

  11. A comparison of three pH control methods for revealing effects of undissociated butyric acid on specific butanol production rate in batch fermentation of Clostridium acetobutylicum

    PubMed Central

    2013-01-01

    pH control has been essential for butanol production with Clostridium acetobutylicum. However, it is not very clear at what pH level the acid crash will occur, at what pH level butanol production will be dominant, and at what pH level butyric acid production will be prevailing. Furthermore, contradictory results have been reported about required acidic conditions for initiation of solventogenesis. In this study, with the aim of further understanding the role of undissociated butyric acid in butanol production, we investigated the correlation between undissociated butyric acid concentration and specific butanol production rate in batch fermentation of Clostridium acetobutylicum by comparing three pH control approaches: NaOH neutralization (at 12, 24 or 36 h), CaCO3 supplementation (2, 5, or 8 g/l) and NaOAc buffering (pH 4.6, 5.0 or 5.6). By neutralizing the fermentation pH to ~5.0 at different time, we observed that neutralization should take place at the beginning of exponential phase (12 h), and otherwise resulting in lower concentrations of undissociated butyric acid, cell biomass and final butanol. CaCO3 supplementation extended cell growth to 36 h and resulted in higher butyrate yield under 8 g/L of CaCO3. In the NaOAc buffering, the highest specific butanol rate (0.58 h−1) was associated with the highest undissociated butyric acid (1.92 g/L). The linear correlation of the undissociated butyric acid with the specific butanol production rates suggested the undissociated butyric acid could be the major driving force for butanol production. PMID:23294525

  12. PH BUFFERING IN FOREST SOIL ORGANIC HORIZONS: RELEVANCE TO ACID PRECIPITATION

    EPA Science Inventory

    Samples of organic surface horizons (Oi, Oe, Oa) from New York State forest soils were equilibrated with 0 to 20 cmol HNO3 Kg(-1) soil in the laboratory by a batch technique designed to simulate reactions of acid precipitation with forest floors. Each organic horizon retained a c...

  13. ALUMINUM SOLUBILITY, CALCIUM-ALUMINUM EXCHANGE, AND PH IN ACID FOREST SOILS

    EPA Science Inventory

    Important components in several models designed to describe the effects of acid deposition on soils and surface waters are the pH-A1 and Ca-A1 exchange relationships. f A1 solubility is controlled by A1 trihydroxide minerals, the theoretical pH-A1 relationship can be described by...

  14. Aggregation and disaggregation of ZnO nanoparticles: influence of pH and adsorption of Suwannee River humic acid.

    PubMed

    Mohd Omar, Fatehah; Abdul Aziz, Hamidi; Stoll, Serge

    2014-01-15

    The surface charge and average size of manufactured ZnO nanoparticles (NPs) were studied as a function of pH to understand the aggregation behavior and importance of the electrostatic interactions in solution. The interactions between ZnO and Suwannee River humic acid (SRHA) were then investigated under a range of environmentally relevant conditions with the ZnO nanoparticles pHPZC as the point of reference. The anionic charges carried by aquatic humic substances were found to play a major role in the aggregation and disaggregation of ZnO nanoparticles. At low concentrations of SRHA (<0.05 mg/L) and below the pHPZC, anionic SRHA was rapidly adsorbed onto the positively charged ZnO NPs hence promoting aggregation. With similar SHRA concentrations, at pHPZC, SRHA was able to control the suspension behavior of the ZnO and promote partial disaggregation in small volumes. This was more distinguishable when the pH was greater than pHPZC as SRHA formed a surface coating on the ZnO nanoparticles and enhanced stability via electrostatic and steric interactions. In most cases, the NP coating by SRHA induced disaggregation behavior in the ZnO nanoparticles and decreased the aggregate size in parallel to increasing SRHA concentrations. Results also suggest that environmental aquatic concentration ranges of humic acids largely modify the stability of aggregated or dispersed ZnO nanoparticles. PMID:24029691

  15. High-rate volatile fatty acid (VFA) production by a granular sludge process at low pH.

    PubMed

    Tamis, J; Joosse, B M; Loosdrecht, M C M van; Kleerebezem, R

    2015-11-01

    Volatile fatty acids (VFA) are proposed platform molecules for the production of basic chemicals and polymers from organic waste streams. In this study we developed a granular sludge process to produce VFA at high rate, yield and purity while minimizing potential operational costs. A lab-scale anaerobic sequencing batch reactor (ASBR) was fed with 10 g l(-1) glucose as model substrate. Inclusion of a short (2 min) settling phase before effluent discharge enabled effective granulation and very high volumetric conversion rates of 150-300 gCOD l(-1)  d(-1) were observed during glucose conversion. The product spectrum remained similar at the tested pH range with acetate and butyrate as the main products, and a total VFA yield of 60-70% on chemical oxygen demand (COD) basis. The requirement for base addition for pH regulation could be reduced from 1.1 to 0.6 mol OH(-) (mol glucose)(-1) by lowering the pH from 5.5 to 4.5. Solids concentrations in the effluent were 0.6 ± 0.3 g l(-1) but could be reduced to 0.02 ± 0.01 g l(-1) by introduction of an additional settling period of 5 min. The efficient production of VFA at low pH with a virtually solid-free effluent increases the economic feasibility of waste-based chemicals and polymer production. Biotechnol. PMID:25950759

  16. Effect of salicylic acid upon trace-metal sorption (Cd, Zn, Co, and Mn) onto alumina, silica, and kaolinite as a function of pH

    SciTech Connect

    Benyahya, L.; Garnier, J.M.

    1999-05-01

    The sorption of four trace metals (Cd, Zn, Co, and Mn) onto alumina, silica, and kaolinite, in the presence or absence of salicylic acid was investigated in batch experiments in the pH range from 4 to 9. The sorption was interpreted in terms of surface complexation using the diffuse layer model (DLM). Equilibrium parameters were optimized using the FITEQL program. The salicylic acid was only significantly sorbed onto the alumina and the sorption was modeled using the anionic monodentate surface complex. In the absence of salicylic acid, the sorption of the trace metals presented different pH edge behaviors, depending on the substrate. Using the cationic monodendate surface complex, the model fitted the experimental data well. In the presence of salicylic acid, at a given pH and depending on the substrate, the sorption of metals was (1) increased, suggesting the occurrence of ternary complexes; (2) reduced (sometimes totally inhibited), due to the complexation with dissolved salicylic acid; or (3) very weakly changed in terms of net effect compared to free-organic-ligand systems. Modeling of the trace-metal sorption in the presence of salicylic acid was performed using ternary surface complexes. In the acidic pH range, this allowed the experimental data to be simulated, but in the alkaline pH range, the model failed to simulate the decrease in sorption. Probable causes of the discrepancies between the experimental data and modeling results are discussed.

  17. Impairment of ascorbic acid's anti-oxidant properties in confined media: inter and intramolecular reactions with air and vanadate at acidic pH.

    PubMed

    Crans, Debbie C; Baruah, Bharat; Gaidamauskas, Ernestas; Lemons, Brant G; Lorenz, Bret B; Johnson, Michael D

    2008-01-01

    The anti-oxidant properties of L-ascorbic acid were investigated in the confined medium produced by a sodium bis(2-ethylhexyl)sulfosuccinate (aerosol-OT, AOT) self-assembled reverse micelle. Using 1H-1H NOESY (proton-proton 2D nuclear overhauser enhancement correlation spectroscopy) NMR spectroscopy, the location of ascorbic acid was investigated and found to be at the AOT-interface in contrast to earlier studies where the ascorbate was assumed to be in the water pool in these microemulsions. The reaction of ascorbic acid with oxygen was investigated using EPR spectroscopy. A delocalized monoanionic ascorbate radical was observed in microemulsions prepared from pH 5.6 stock solutions. This is in contrast to studies carried out in aqueous media where no radical formation was observed. The oxidation of ascorbic acid by aqueous V(V) was investigated in reverse micelles. Modest changes in the kinetic parameters were observed for this system compared to that in water. Details of these reactions were examined and can be summarized as the microemulsion solvating and stabilizing reactive intermediates via rate inhibition or enhancement. The inhibition of the oxidation is due to solvation stabilization of ascorbic acid in microemulsion media. Since ascorbate is a valuable marker of oxidative stress, our results suggest that compartmentization can modify the stabilization of the ascorbate radical and the changes in properties could be important in biological systems. PMID:18331759

  18. Rumen morphometrics and the effect of digesta pH and volume on volatile fatty acid absorption.

    PubMed

    Melo, L Q; Costa, S F; Lopes, F; Guerreiro, M C; Armentano, L E; Pereira, M N

    2013-04-01

    The effects of rumen digesta volume and pH on VFA absorption and its relation to rumen wall morphology were evaluated. Nine rumen cannulated cows formed 3 groups based on desired variation in rumen morphology: The High group was formed by Holsteins yielding 25.9 kg milk/d and fed on a high-grain total