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Sample records for acid pka values

  1. Rationalization of the pKa values of alcohols and thiols using atomic charge descriptors and its application to the prediction of amino acid pKa's.

    PubMed

    Ugur, Ilke; Marion, Antoine; Parant, Stéphane; Jensen, Jan H; Monard, Gerald

    2014-08-25

    In a first step toward the development of an efficient and accurate protocol to estimate amino acids' pKa's in proteins, we present in this work how to reproduce the pKa's of alcohol and thiol based residues (namely tyrosine, serine, and cysteine) in aqueous solution from the knowledge of the experimental pKa's of phenols, alcohols, and thiols. Our protocol is based on the linear relationship between computed atomic charges of the anionic form of the molecules (being either phenolates, alkoxides, or thiolates) and their respective experimental pKa values. It is tested with different environment approaches (gas phase or continuum solvent-based approaches), with five distinct atomic charge models (Mulliken, Löwdin, NPA, Merz-Kollman, and CHelpG), and with nine different DFT functionals combined with 16 different basis sets. Moreover, the capability of semiempirical methods (AM1, RM1, PM3, and PM6) to also predict pKa's of thiols, phenols, and alcohols is analyzed. From our benchmarks, the best combination to reproduce experimental pKa's is to compute NPA atomic charge using the CPCM model at the B3LYP/3-21G and M062X/6-311G levels for alcohols (R(2) = 0.995) and thiols (R(2) = 0.986), respectively. The applicability of the suggested protocol is tested with tyrosine and cysteine amino acids, and precise pKa predictions are obtained. The stability of the amino acid pKa's with respect to geometrical changes is also tested by MM-MD and DFT-MD calculations. Considering its strong accuracy and its high computational efficiency, these pKa prediction calculations using atomic charges indicate a promising method for predicting amino acids' pKa in a protein environment.

  2. Rationalization of the pKa values of alcohols and thiols using atomic charge descriptors and its application to the prediction of amino acid pKa's.

    PubMed

    Ugur, Ilke; Marion, Antoine; Parant, Stéphane; Jensen, Jan H; Monard, Gerald

    2014-08-25

    In a first step toward the development of an efficient and accurate protocol to estimate amino acids' pKa's in proteins, we present in this work how to reproduce the pKa's of alcohol and thiol based residues (namely tyrosine, serine, and cysteine) in aqueous solution from the knowledge of the experimental pKa's of phenols, alcohols, and thiols. Our protocol is based on the linear relationship between computed atomic charges of the anionic form of the molecules (being either phenolates, alkoxides, or thiolates) and their respective experimental pKa values. It is tested with different environment approaches (gas phase or continuum solvent-based approaches), with five distinct atomic charge models (Mulliken, Löwdin, NPA, Merz-Kollman, and CHelpG), and with nine different DFT functionals combined with 16 different basis sets. Moreover, the capability of semiempirical methods (AM1, RM1, PM3, and PM6) to also predict pKa's of thiols, phenols, and alcohols is analyzed. From our benchmarks, the best combination to reproduce experimental pKa's is to compute NPA atomic charge using the CPCM model at the B3LYP/3-21G and M062X/6-311G levels for alcohols (R(2) = 0.995) and thiols (R(2) = 0.986), respectively. The applicability of the suggested protocol is tested with tyrosine and cysteine amino acids, and precise pKa predictions are obtained. The stability of the amino acid pKa's with respect to geometrical changes is also tested by MM-MD and DFT-MD calculations. Considering its strong accuracy and its high computational efficiency, these pKa prediction calculations using atomic charges indicate a promising method for predicting amino acids' pKa in a protein environment. PMID:25089727

  3. Estimated pKa values for the environmentally relevant C1 through C8 perfluorinated sulfonic acid isomers.

    PubMed

    Rayne, Sierra; Forest, Kaya

    2016-10-14

    In order to estimate isomer-specific acidity constants (pKa) for the perfluorinated sulfonic acid (PFSA) environmental contaminants, the parameterization method 6 (PM6) pKa prediction method was extensively validated against a wide range of carbon oxyacids and related sulfonic/sulfinic acids. Excellent pKa prediction performance was observed for the carbon oxyacids using the PM6 method, but this approach was found to have a severe positive bias for sulfonic/sulfinic acids. To overcome this obstacle, a correlation was developed between non-adjusted PM6 pKa values and the corresponding experimentally obtained/estimated acidity constants for a range of representative alkyl, aryl and halogen-substituted sulfonic acids. Application of this correction to the PM6 values allows for extension of this computational method to a new acid functional group. When used to estimate isomer-specific pKa values for the C1 through C8 PFSAs, the modified PM6 approach suggests an adjusted pKa range from -5.3 to -9.0, indicating that all members of this class of well-known environmental contaminants will be effectively completely dissociated in aquatic systems.

  4. Estimated pKa values for the environmentally relevant C1 through C8 perfluorinated sulfonic acid isomers.

    PubMed

    Rayne, Sierra; Forest, Kaya

    2016-10-14

    In order to estimate isomer-specific acidity constants (pKa) for the perfluorinated sulfonic acid (PFSA) environmental contaminants, the parameterization method 6 (PM6) pKa prediction method was extensively validated against a wide range of carbon oxyacids and related sulfonic/sulfinic acids. Excellent pKa prediction performance was observed for the carbon oxyacids using the PM6 method, but this approach was found to have a severe positive bias for sulfonic/sulfinic acids. To overcome this obstacle, a correlation was developed between non-adjusted PM6 pKa values and the corresponding experimentally obtained/estimated acidity constants for a range of representative alkyl, aryl and halogen-substituted sulfonic acids. Application of this correction to the PM6 values allows for extension of this computational method to a new acid functional group. When used to estimate isomer-specific pKa values for the C1 through C8 PFSAs, the modified PM6 approach suggests an adjusted pKa range from -5.3 to -9.0, indicating that all members of this class of well-known environmental contaminants will be effectively completely dissociated in aquatic systems. PMID:27389973

  5. Ab Initio Molecular Dynamics Simulations of Amino Acids in Aqueous Solutions: Estimating pKa Values from Metadynamics Sampling.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2015-09-17

    Changes in the protonation and deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. Here, we report calculations of the free-energy profile for the protonation-deprotonation reaction of the 20 canonical α amino acids in aqueous solutions using ab initio Car-Parrinello molecular dynamics simulations coupled with metadynamics sampling. We show here that the calculated change in free energy of the dissociation reaction provides estimates of the multiple pKa values of the amino acids that are in good agreement with experiment. We use the bond-length-dependent number of the protons coordinated to the hydroxyl oxygen of the carboxylic and the amine groups as the collective variables to explore the free-energy profiles of the Bronsted acid-base chemistry of amino acids in aqueous solutions. We ensure that the amino acid undergoing dissociation is solvated by at least three hydrations shells with all water molecules included in the simulations. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error, with respect to experimental results, of 0.2 pKa units. PMID:26331783

  6. Ab Initio Molecular Dynamics Simulations of Amino Acids in Aqueous Solutions: Estimating pKa Values from Metadynamics Sampling.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2015-09-17

    Changes in the protonation and deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. Here, we report calculations of the free-energy profile for the protonation-deprotonation reaction of the 20 canonical α amino acids in aqueous solutions using ab initio Car-Parrinello molecular dynamics simulations coupled with metadynamics sampling. We show here that the calculated change in free energy of the dissociation reaction provides estimates of the multiple pKa values of the amino acids that are in good agreement with experiment. We use the bond-length-dependent number of the protons coordinated to the hydroxyl oxygen of the carboxylic and the amine groups as the collective variables to explore the free-energy profiles of the Bronsted acid-base chemistry of amino acids in aqueous solutions. We ensure that the amino acid undergoing dissociation is solvated by at least three hydrations shells with all water molecules included in the simulations. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error, with respect to experimental results, of 0.2 pKa units.

  7. Estimating successive pKa values of polyprotic acids from ab initio molecular dynamics using metadynamics: the dissociation of phthalic acid and its isomers.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2015-03-01

    Estimation of the dissociation constant, or pKa, of weak acids continues to be a central goal in theoretical chemistry. Here we show that ab initio Car-Parrinello molecular dynamics simulations in conjunction with metadynamics calculations of the free energy profile of the dissociation reaction can provide reasonable estimates of the successive pKa values of polyprotic acids. We use the distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic group as the collective variable to explore the free energy profile of the dissociation process. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule, were included explicitly in the computation procedure. Two distinct minima corresponding to the dissociated and un-dissociated states of the acid are observed and the difference in their free energy values provides the estimate for pKa, the acid dissociation constant. We show that the method predicts the pKa value of benzoic acid in good agreement with experiment and then show using phthalic acid (benzene dicarboxylic acid) as a test system that both the first and second pKa values as well, as the subtle difference in their values for different isomers can be predicted in reasonable agreement with experimental data. PMID:25652329

  8. Estimating successive pKa values of polyprotic acids from ab initio molecular dynamics using metadynamics: the dissociation of phthalic acid and its isomers.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2015-03-01

    Estimation of the dissociation constant, or pKa, of weak acids continues to be a central goal in theoretical chemistry. Here we show that ab initio Car-Parrinello molecular dynamics simulations in conjunction with metadynamics calculations of the free energy profile of the dissociation reaction can provide reasonable estimates of the successive pKa values of polyprotic acids. We use the distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic group as the collective variable to explore the free energy profile of the dissociation process. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule, were included explicitly in the computation procedure. Two distinct minima corresponding to the dissociated and un-dissociated states of the acid are observed and the difference in their free energy values provides the estimate for pKa, the acid dissociation constant. We show that the method predicts the pKa value of benzoic acid in good agreement with experiment and then show using phthalic acid (benzene dicarboxylic acid) as a test system that both the first and second pKa values as well, as the subtle difference in their values for different isomers can be predicted in reasonable agreement with experimental data.

  9. Acidity constants in methanol/water mixtures of polycarboxylic acids used in drug salt preparations. Potentiometric determination of aqueous pKa values of quetiapine formulated as hemifumarate.

    PubMed

    Garrido, Gemma; Ràfols, Clara; Bosch, Elisabeth

    2006-05-01

    The acidic dissociation constants in a number of methanol/water mixtures of mono and polycarboxylic acids commonly used in the preparation of drug salts were determined. These solvent mixtures are usually used to determine the pKa of drugs of low aqueous solubility. However, when these drugs are prepared in salt form, the acid-base equilibria of both the basic drug and the counter-anion are involved in the potentiometric titration curves. In these instances, the inclusion of the pKa of acids as constant values in the curve fitting provides easy computation of the drug pKa without the need of any previous step to get the free base. As an application example, the aqueous pKa values of the quetiapine formulated as hemifumarate (Seroquel) were estimated by extrapolation from the experimental pKa in several methanol/water mixtures, which were then calculated according to the suitable constants of fumaric acid. The estimated aqueous pKa values of quetiapine are compared with those directly obtained in aqueous solution by potentiometry and by capillary electrophoresis.

  10. High acidity tolerance in lichens with fumarprotocetraric, perlatolic or thamnolic acids is correlated with low pKa1 values of these lichen substances.

    PubMed

    Hauck, Markus; Jürgens, Sascha-René; Huneck, Siegfried; Leuschner, Christoph

    2009-10-01

    The depsidone fumarprotocetraric acid as well as the depsides perlatolic and thamnolic acids are lichen secondary metabolites. Their first dissociation constants (pK(a1)) in methanol were determined to be 2.7 for perlatolic acid and 2.8 for fumarprotocetraric and thamnolic acids by UV spectroscopy. Lower pK(a1) values are, so far, not known from lichen substances. Several lichens producing at least one of these compounds are known for their outstanding tolerance to acidic air pollution. This is demonstrated by evaluating published pH preferences for central European lichens. The low pK(a1) values suggest that strong dissociation of the studied lichen substances is a prerequisite for the occurrence of lichens with these compounds on very acidic substrata, as protonated lichen substances of different chemical groups, but not their conjugated bases, are known to shuttle protons into the cytoplasm and thereby apparently damage lichens.

  11. Dissociation constants of weak acids from ab initio molecular dynamics using metadynamics: influence of the inductive effect and hydrogen bonding on pKa values.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2014-11-26

    The theoretical estimation of the dissociation constant, or pKa, of weak acids continues to be a challenging field. Here, we show that ab initio Car-Parrinello molecular dynamics simulations in conjunction with metadynamics calculations of the free-energy profile of the dissociation reaction provide reasonable estimates of the pKa value. Water molecules, sufficient to complete the three hydration shells surrounding the acid molecule, were included explicitly in the computation procedure. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid, and the difference in their values provides the estimate for pKa. We show for a series of organic acids that CPMD simulations in conjunction with metadynamics can provide reasonable estimates of pKa values. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic acid, and the isomers of hydroxybenzoic acid. These systems were chosen to highlight that the procedure could correctly account for the influence of the inductive effect as well as hydrogen bonding on pKa values of weak organic acids. In both situations, the CPMD metadynamics procedure faithfully reproduces the experimentally observed trend and the magnitudes of the pKa values.

  12. First Principles Calculations of Aqueous pKa Values for Organic and Inorganic Acids Using COSMO-RS Reveal an Inconsistency in the Slope of the pKa Scale.

    PubMed

    Klamt, Andreas; Eckert, Frank; Diedenhofen, Michael; Beck, Michael E

    2003-11-01

    The COSMO-RS method, a combination of the quantum chemical dielectric continuum solvation model COSMO with a statistical thermodynamics treatment for more realistic solvation (RS) simulations, has been used for the direct prediction of pKa constants of a large variety of 64 organic and inorganic acids. A highly significant correlation of r(2) = 0.984 with a standard deviation of only 0.49 between the calculated values of the free energies of dissociation and the experimental pKa values was found, without any special adjustment of the method. Thus, we have a theoretical a priori prediction method for pKa, which has the regression constant and the slope as only adjusted parameters. Such a method can be of great value in many areas of physical chemistry, especially in pharmaceutical and agrochemical industry. To our surprise, the slope of pKa vs ΔGdiss is only 58% of the theoretically expected value of 1/RTln(10). A careful analysis with respect to different contributions as well as a comparison with the work of other authors excludes the possibility that the discrepancy is due to weaknesses of the calculation method. Hence, we must conclude that the experimental pKa scale depends differently on the free energy of dissociation than generally assumed. PMID:26313337

  13. Automated determination of the pKa values of 4-hydroxybenzoic acid in cosolvent-water mixtures and related solvent effects using a modified HPLC system.

    PubMed

    Miklautz, H; Keller, D; Lopez Holguin, F; Woloszczak, R

    2006-03-01

    An automated spectrophotometric method based on an HPLC system with a diode array detector was used to determine the pK(a) values of compounds with low water solubility in a universal buffer containing acetonitrile as cosolvent. The column of the system was replaced with a capillary connecting the injection system and the diode array detector. Specific solvent effects were corrected for using the dielectric constants of the mixed solvent and pure water. The method was tested using 4-hydroxybenzoic acid and the results were compared with those obtained with a spectrophotometer. Linear regression lines with different slopes were obtained from spectrophotometric measurements of different cosolvent-water mixtures. These effects were shown to depend upon the polarity of the solvent-water mixture, and they were explained by the solvatochromic behavior of the 4-hydroxybenzoic acid in the solvent-water mixture.

  14. Bayesian model aggregation for ensemble-based estimates of protein pKa values

    SciTech Connect

    Gosink, Luke J.; Hogan, Emilie A.; Pulsipher, Trenton C.; Baker, Nathan A.

    2014-03-01

    This paper investigates an ensemble-based technique called Bayesian Model Averaging (BMA) to improve the performance of protein amino acid p$K_a$ predictions. Structure-based p$K_a$ calculations play an important role in the mechanistic interpretation of protein structure and are also used to determine a wide range of protein properties. A diverse set of methods currently exist for p$K_a$ prediction, ranging from empirical statistical models to {\\it ab initio} quantum mechanical approaches. However, each of these methods are based on a set of assumptions that have inherent bias and sensitivities that can effect a model's accuracy and generalizability for p$K_a$ prediction in complicated biomolecular systems. We use BMA to combine eleven diverse prediction methods that each estimate pKa values of amino acids in staphylococcal nuclease. These methods are based on work conducted for the pKa Cooperative and the pKa measurements are based on experimental work conducted by the Garc{\\'i}a-Moreno lab. Our study demonstrates that the aggregated estimate obtained from BMA outperforms all individual prediction methods in our cross-validation study with improvements from 40-70\\% over other method classes. This work illustrates a new possible mechanism for improving the accuracy of p$K_a$ prediction and lays the foundation for future work on aggregate models that balance computational cost with prediction accuracy.

  15. Cysteine pK(a) values for the bacterial peroxiredoxin AhpC.

    PubMed

    Nelson, Kimberly J; Parsonage, Derek; Hall, Andrea; Karplus, P Andrew; Poole, Leslie B

    2008-12-01

    Salmonella typhimurium AhpC is a founding member of the peroxiredoxin family, a ubiquitous group of cysteine-based peroxidases with high reactivity toward hydrogen peroxide, organic hydroperoxides, and peroxynitrite. For all of the peroxiredoxins, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), acts as a nucleophile in attacking the peroxide substrate, forming a cysteine sulfenic acid at the active site. Because thiolates are far stronger nucleophiles than thiol groups, it is generally accepted that cysteine-based peroxidases should exhibit pK(a) values lower than an unperturbed value of 8.3-8.5. In this investigation, several independent approaches were used to assess the pK(a) of the two cysteinyl residues of AhpC. Methods using two different iodoacetamide derivatives yielded unperturbed pK(a) values (7.9-8.7) for both cysteines, apparently due to reactivity with the wrong conformation of C(P) (i.e., locally unfolded and flipped out of the active site), as supported by X-ray crystallographic analyses. A functional pK(a) of 5.94 +/- 0.10 presumably reflecting the titration of C(P) within the fully folded active site was obtained by measuring AhpC competition with horseradish peroxidase for hydrogen peroxide; this value is quite similar to that obtained by analyzing the pH dependence of the epsilon(240) of wild-type AhpC (5.84 +/- 0.02) and similar to those obtained for two typical 2-cysteine peroxiredoxins from Saccharomyces cerevisiae (5.4 and 6.0). Thus, the pK(a) value of AhpC balances the need for a deprotonated thiol (at pH 7, approximately 90% of the C(P) would be deprotonated) with the fact that thiolates with higher pK(a) values are stronger nucleophiles. PMID:18986167

  16. Assigning the pKa's of Polyprotic Acids.

    ERIC Educational Resources Information Center

    Bodner, George M.

    1986-01-01

    Discusses (1) polyproptic acids for which the difference between K-a's is large; (2) the Henderson-Hasselbach equation; (3) polyprotic acids for which the difference between K-a's is small; (4) analysis of microscopic dissociation constants for cysteine; and (5) analysis of pK-a data. (JN)

  17. Theoretical study of firefly luciferin pKa values--relative absorption intensity in aqueous solutions.

    PubMed

    Hiyama, Miyabi; Akiyama, Hidefumi; Yamada, Kenta; Koga, Nobuaki

    2013-01-01

    Ground-state vibrational analyses of firefly luciferin and its conjugate acids and bases are performed. The Gibbs free energies obtained from these analyses are used to estimate pKa values for phenolic hydroxy and carboxy groups and the N-H(+) bond in the N-protonated thiazoline or benzothiazole ring of firefly luciferin. The theoretical pKa values are corrected using the experimental values. The concentrations of these chemical species in solutions with different pH values are estimated from their corrected pKa values, and the pH dependence of their relative absorption intensities is elucidated. With the results obtained we assign the experimental spectra unequivocally. Especially, the small peak near 400 nm at pH 1-2 in experimental absorption spectra is clarified to be due to the excitation of carboxylate anion with N-protonated thiazoline ring of firefly luciferin. Our results show that the pKa values of chemical species, which are contained in the aqueous solutions, are effective to assign experimental absorption spectra. PMID:23360188

  18. Theoretical study of firefly luciferin pKa values--relative absorption intensity in aqueous solutions.

    PubMed

    Hiyama, Miyabi; Akiyama, Hidefumi; Yamada, Kenta; Koga, Nobuaki

    2013-01-01

    Ground-state vibrational analyses of firefly luciferin and its conjugate acids and bases are performed. The Gibbs free energies obtained from these analyses are used to estimate pKa values for phenolic hydroxy and carboxy groups and the N-H(+) bond in the N-protonated thiazoline or benzothiazole ring of firefly luciferin. The theoretical pKa values are corrected using the experimental values. The concentrations of these chemical species in solutions with different pH values are estimated from their corrected pKa values, and the pH dependence of their relative absorption intensities is elucidated. With the results obtained we assign the experimental spectra unequivocally. Especially, the small peak near 400 nm at pH 1-2 in experimental absorption spectra is clarified to be due to the excitation of carboxylate anion with N-protonated thiazoline ring of firefly luciferin. Our results show that the pKa values of chemical species, which are contained in the aqueous solutions, are effective to assign experimental absorption spectra.

  19. Isodesmic reaction for accurate theoretical pKa calculations of amino acids and peptides.

    PubMed

    Sastre, S; Casasnovas, R; Muñoz, F; Frau, J

    2016-04-28

    Theoretical and quantitative prediction of pKa values at low computational cost is a current challenge in computational chemistry. We report that the isodesmic reaction scheme provides semi-quantitative predictions (i.e. mean absolute errors of 0.5-1.0 pKa unit) for the pKa1 (α-carboxyl), pKa2 (α-amino) and pKa3 (sidechain groups) of a broad set of amino acids and peptides. This method fills the gaps of thermodynamic cycles for the computational pKa calculation of molecules that are unstable in the gas phase or undergo proton transfer reactions or large conformational changes from solution to the gas phase. We also report the key criteria to choose a reference species to make accurate predictions. This method is computationally inexpensive and makes use of standard density functional theory (DFT) and continuum solvent models. It is also conceptually simple and easy to use for researchers not specialized in theoretical chemistry methods. PMID:27052591

  20. Determination of pKa values for deprotonable nucleobases in short model oligonucleotides.

    PubMed

    González-Olvera, Julio C; Martínez-Reyes, José; González-Jasso, Eva; Pless, Reynaldo C

    2015-11-01

    The deprotonation of ionizable nucleobases centrally placed in short model oligonucleotides was examined under different physical conditions, using UV absorption spectroscopy. The oligonucleotide sequences were designed so that only the central base would be ionized over the pH range examined. pKa values of 9.90±0.01 and 9.34±0.04 were determined for the guanine group in the oligomer d-ACAGCAC and 2'-deoxyguanosine, respectively, both at 25°C and 0.1M NaCl. Lengthening the oligonucleotide up to the tridecamer stage further increases the pKa of the central guanine moiety. Electrolyte concentration, temperature, and mixed water-ethanol solvents affect the acidity of the central base. Changes in the sequence surrounding the central guanine can also have a significant effect, especially in the case of strongly stacking sequences. The pKa values were also determined for the hepta(2'-O-methyl)ribonucleotide and the heptamer PNA of identical sequence, as well as for oligodeoxyribonucleotides with different deprotonable bases, viz. thymine, uracil, or hypoxanthine, in the central position. The results are interpreted in terms of the electric-field effect exerted on the departing proton by the negative electric charges located on the internucleotide phosphate groups, and calculations show this effect to approximately explain the magnitude of the pKa difference observed between the deoxyriboheptanucleotide and its electroneutral PNA analogue. PMID:26188860

  1. Prediction of pKa values using the PM6 semiempirical method

    PubMed Central

    Kromann, Jimmy C.; Larsen, Frej; Moustafa, Hadeel

    2016-01-01

    The PM6 semiempirical method and the dispersion and hydrogen bond-corrected PM6-D3H+ method are used together with the SMD and COSMO continuum solvation models to predict pKa values of pyridines, alcohols, phenols, benzoic acids, carboxylic acids, and phenols using isodesmic reactions and compared to published ab initio results. The pKa values of pyridines, alcohols, phenols, and benzoic acids considered in this study can generally be predicted with PM6 and ab initio methods to within the same overall accuracy, with average mean absolute differences (MADs) of 0.6–0.7 pH units. For carboxylic acids, the accuracy (0.7–1.0 pH units) is also comparable to ab initio results if a single outlier is removed. For primary, secondary, and tertiary amines the accuracy is, respectively, similar (0.5–0.6), slightly worse (0.5–1.0), and worse (1.0–2.5), provided that di- and tri-ethylamine are used as reference molecules for secondary and tertiary amines. When applied to a drug-like molecule where an empirical pKa predictor exhibits a large (4.9 pH unit) error, we find that the errors for PM6-based predictions are roughly the same in magnitude but opposite in sign. As a result, most of the PM6-based methods predict the correct protonation state at physiological pH, while the empirical predictor does not. The computational cost is around 2–5 min per conformer per core processor, making PM6-based pKa prediction computationally efficient enough to be used for high-throughput screening using on the order of 100 core processors.

  2. Prediction of pKa values using the PM6 semiempirical method.

    PubMed

    Kromann, Jimmy C; Larsen, Frej; Moustafa, Hadeel; Jensen, Jan H

    2016-01-01

    The PM6 semiempirical method and the dispersion and hydrogen bond-corrected PM6-D3H+ method are used together with the SMD and COSMO continuum solvation models to predict pKa values of pyridines, alcohols, phenols, benzoic acids, carboxylic acids, and phenols using isodesmic reactions and compared to published ab initio results. The pKa values of pyridines, alcohols, phenols, and benzoic acids considered in this study can generally be predicted with PM6 and ab initio methods to within the same overall accuracy, with average mean absolute differences (MADs) of 0.6-0.7 pH units. For carboxylic acids, the accuracy (0.7-1.0 pH units) is also comparable to ab initio results if a single outlier is removed. For primary, secondary, and tertiary amines the accuracy is, respectively, similar (0.5-0.6), slightly worse (0.5-1.0), and worse (1.0-2.5), provided that di- and tri-ethylamine are used as reference molecules for secondary and tertiary amines. When applied to a drug-like molecule where an empirical pKa predictor exhibits a large (4.9 pH unit) error, we find that the errors for PM6-based predictions are roughly the same in magnitude but opposite in sign. As a result, most of the PM6-based methods predict the correct protonation state at physiological pH, while the empirical predictor does not. The computational cost is around 2-5 min per conformer per core processor, making PM6-based pKa prediction computationally efficient enough to be used for high-throughput screening using on the order of 100 core processors. PMID:27602298

  3. Prediction of pKa values using the PM6 semiempirical method

    PubMed Central

    Kromann, Jimmy C.; Larsen, Frej; Moustafa, Hadeel

    2016-01-01

    The PM6 semiempirical method and the dispersion and hydrogen bond-corrected PM6-D3H+ method are used together with the SMD and COSMO continuum solvation models to predict pKa values of pyridines, alcohols, phenols, benzoic acids, carboxylic acids, and phenols using isodesmic reactions and compared to published ab initio results. The pKa values of pyridines, alcohols, phenols, and benzoic acids considered in this study can generally be predicted with PM6 and ab initio methods to within the same overall accuracy, with average mean absolute differences (MADs) of 0.6–0.7 pH units. For carboxylic acids, the accuracy (0.7–1.0 pH units) is also comparable to ab initio results if a single outlier is removed. For primary, secondary, and tertiary amines the accuracy is, respectively, similar (0.5–0.6), slightly worse (0.5–1.0), and worse (1.0–2.5), provided that di- and tri-ethylamine are used as reference molecules for secondary and tertiary amines. When applied to a drug-like molecule where an empirical pKa predictor exhibits a large (4.9 pH unit) error, we find that the errors for PM6-based predictions are roughly the same in magnitude but opposite in sign. As a result, most of the PM6-based methods predict the correct protonation state at physiological pH, while the empirical predictor does not. The computational cost is around 2–5 min per conformer per core processor, making PM6-based pKa prediction computationally efficient enough to be used for high-throughput screening using on the order of 100 core processors. PMID:27602298

  4. Predicting the pKa and stability of organic acids and bases at an oil-water interface.

    PubMed

    Andersson, M P; Olsson, M H M; Stipp, S L S

    2014-06-10

    We have used density functional theory and the implicit solvent model, COSMO-RS, to investigate how the acidity constant, pKa, of organic acids and bases adsorbed at the organic compound-aqueous solution interface changes, compared to its value in the aqueous phase. The pKa determine the surface charge density of the molecules that accumulate at the fluid-fluid interface. We have estimated the pKa by comparing the stability of the protonated and unprotonated forms of a series of molecules in the bulk aqueous solution and at an interface where parts of each molecule reside in the hydrophobic phase and the rest remains in the hydrophilic phase. We found that the pKa for acids is shifted by ∼1 pH unit to higher values compared to the bulk water pKa, whereas they are shifted to lower values by a similar amount for bases. Because this pKa shift is similar in magnitude for each of the molecules studied, we propose that the pKa for molecules at a water-organic compound interface can easily be predicted by adding a small shift to the aqueous pKa. This shift is general and correlates with the functional group. We also found that the relative composition of molecules at the fluid-fluid interface is not the same as in the bulk. For example, species such as carboxylic acids are enriched at the interface, where they can dominate surface properties, even when they are a modest component in the bulk fluid. For high surface concentrations of carboxylic acid groups at an interface, such as a self-assembled monolayer, we have demonstrated that the pKa depends on the degree of deprotonation through direct hydrogen bonding between protonated and deprotonated acidic headgroups.

  5. The Determination of "Apparent" pKa's. Part II: An Experiment Using Very Weak Acids (pKa's > 11.4).

    ERIC Educational Resources Information Center

    Cawley, John J.

    1995-01-01

    Presents an experiment designed to show students that the Henderson-Hasselbalch equation will fail when they use this particular one-half titration technique for acids with large pKa's. Involves determining the apparent pKa for such acids and using that to calculate the true pKa. (JRH)

  6. Accurate pKa calculation of the conjugate acids of alkanolamines, alkaloids and nucleotide bases by quantum chemical methods.

    PubMed

    Gangarapu, Satesh; Marcelis, Antonius T M; Zuilhof, Han

    2013-04-01

    The pKa of the conjugate acids of alkanolamines, neurotransmitters, alkaloid drugs and nucleotide bases are calculated with density functional methods (B3LYP, M08-HX and M11-L) and ab initio methods (SCS-MP2, G3). Implicit solvent effects are included with a conductor-like polarizable continuum model (CPCM) and universal solvation models (SMD, SM8). G3, SCS-MP2 and M11-L methods coupled with SMD and SM8 solvation models perform well for alkanolamines with mean unsigned errors below 0.20 pKa units, in all cases. Extending this method to the pKa calculation of 35 nitrogen-containing compounds spanning 12 pKa units showed an excellent correlation between experimental and computational pKa values of these 35 amines with the computationally low-cost SM8/M11-L density functional approach.

  7. Empirically Fitted Parameters for Calculating pKa Values with Small Deviations from Experiments Using a Simple Computational Strategy.

    PubMed

    Galano, Annia; Pérez-González, Adriana; Castañeda-Arriaga, Romina; Muñoz-Rugeles, Leonardo; Mendoza-Sarmiento, Gabriela; Romero-Silva, Antonio; Ibarra-Escutia, Agustin; Rebollar-Zepeda, Aida Mariana; León-Carmona, Jorge Rafael; Hernández-Olivares, Manuel Alejandro; Alvarez-Idaboy, Juan Raúl

    2016-09-26

    Two empirically fitted parameters have been derived for 74 levels of theory. They allow fast and reliable pKa calculations using only the Gibbs energy difference between an acid and its conjugated base in aqueous solution (ΔGs(BA)). The parameters were obtained by least-squares fits of ΔGs(BA) vs experimental pKa values for phenols, carboxylic acids, and amines using training sets of 20 molecules for each chemical family. Test sets of 10 molecules per family-completely independent from the training set-were used to verify the reliability of the fitting parameters method. It was found that, except for MP2, deviations from experiments are lower than 0.5 pKa units. Moreover, mean unsigned errors lower than 0.35 pKa units were found for the 98.6%, 98.6%, and 94.6% of the tested levels of theory for phenols, carboxylic acids and amines, respectively. The parameters estimated here are expected to facilitate computationally based estimations of pKa values of species for which this magnitude is still unknown, with uncertainties similar to the experimental ones. However, the present study deals only with molecules of modest complexity, thus the reliability of the FP method for more complex systems remains to be tested.

  8. The intrinsic pKa values for phosphatidylserine and phosphatidylethanolamine in phosphatidylcholine host bilayers.

    PubMed Central

    Tsui, F C; Ojcius, D M; Hubbell, W L

    1986-01-01

    Potentiometric titrations and surface potential measurements have been used to determine the intrinsic pKa values of both the carboxyl and amino groups of phosphatidylserine (PS) in mixed vesicles of PS and phosphatidylcholine (PC), and also of the amino group of phosphatidylethanolamine (PE) in mixed PE-PC vesicles. The pKa of the carboxyl group of PS in liposomes with different PS/PC lipid ratios measured by the two different methods is 3.6 +/- 0.1, and the pKa of its amino group is 9.8 +/- 0.1. The pKa of the amino group of PE in PE-PC vesicles, determined solely by surface potential measurements, is 9.6 +/- 0.1. These pKa values are independent of the aqueous phase ionic strength and of the effect of the liposome's surface potential due to the presence of these partially charged lipids. PMID:3955180

  9. Application of chemometrics in determination of the acid dissociation constants (pKa) of several benzodiazepine derivatives as poorly soluble drugs in the presence of ionic surfactants.

    PubMed

    Shayesteh, Tavakol Heidary; Radmehr, Moojan; Khajavi, Farzad; Mahjub, Reza

    2015-03-10

    In this study, the acid dissociation constants (pKa) of some benzodiazepine derivatives including chlordiazepoxide, clonazepam, lorazepam, and oxazepam in aqueous micellar solution were determined spectrophotometrically at an ionic strength of 0.1M at 25°C. The effect of cetyl trimethylammonium bromide (CTAB) as a cationic and sodium n-dodecyl sulfate(SDS) as an anionic surfactant on the absorption spectra of benzodiazepine drugs at different pH values were studied. The acidity constants of all related species are estimated by considering the surfactant concept and the application of chemometric methods using the whole spectral fitting of the collected data to an established factor analysis model. DATAN® software (Ver. 5.0, Multid Analyses AB, and Goteborg, Sweden) was applied to determine the acidity constants. In this study, a simple and fast method to determine the ionization constant (pKa) of poorly soluble drugs was developed using surfactants. The acidity constant (i.e. pKa) for chlordiazepoxide, clonazepam, lorazepam, and oxazepam were reported as 4.62, pKa1 value of 1.52 and pKa2 value of 10.51, pKa1 value of 1.53 and pKa2 value of 10.92 and pKa1 value 1.63 and pKa2 value of 11.21 respectively. The results showed that the peak values in the spectrophotometric absorption spectra of drugs are influenced by the presence of anionic and cationic surfactants. According to the results, by changing the SDS concentration from 0 to 0.05M, the pKa of chlordiazepoxide was increased to 5.9, the pKa1 of lorazepam was decreased to 0.1 while the pKa2 was increased to 11.5. Increase in SDS concentration has not shown significant alteration in pKa of clonazepam and oxazepam. Results indicate that by Changing the CTAB concentration from 0 to 0.05M, the pKa of chlordiazepoxide was reduced to 4.4, the pKa1 of clonazepam was decreased to 0.1 and the pKa2 was decreased to 9.1, the pKa1 of lorazepam was decreased to 0.4 and the pKa2 was decreased to 9.4, the pKa1 of oxazepam was

  10. Development of Methods for the Determination of pKa Values

    PubMed Central

    Reijenga, Jetse; van Hoof, Arno; van Loon, Antonie; Teunissen, Bram

    2013-01-01

    The acid dissociation constant (pKa) is among the most frequently used physicochemical parameters, and its determination is of interest to a wide range of research fields. We present a brief introduction on the conceptual development of pKa as a physical parameter and its relationship to the concept of the pH of a solution. This is followed by a general summary of the historical development and current state of the techniques of pKa determination and an attempt to develop insight into future developments. Fourteen methods of determining the acid dissociation constant are placed in context and are critically evaluated to make a fair comparison and to determine their applications in modern chemistry. Additionally, we have studied these techniques in light of present trends in science and technology and attempt to determine how these trends might affect future developments in the field. PMID:23997574

  11. PPD v1.0--an integrated, web-accessible database of experimentally determined protein pKa values.

    PubMed

    Toseland, Christopher P; McSparron, Helen; Davies, Matthew N; Flower, Darren R

    2006-01-01

    The Protein pK(a) Database (PPD) v1.0 provides a compendium of protein residue-specific ionization equilibria (pK(a) values), as collated from the primary literature, in the form of a web-accessible postgreSQL relational database. Ionizable residues play key roles in the molecular mechanisms that underlie many biological phenomena, including protein folding and enzyme catalysis. The PPD serves as a general protein pK(a) archive and as a source of data that allows for the development and improvement of pK(a) prediction systems. The database is accessed through an HTML interface, which offers two fast, efficient search methods: an amino acid-based query and a Basic Local Alignment Search Tool search. Entries also give details of experimental techniques and links to other key databases, such as National Center for Biotechnology Information and the Protein Data Bank, providing the user with considerable background information. The database can be found at the following URL: http://www.jenner.ac.uk/PPD. PMID:16381845

  12. Progress in the prediction of pKa values in proteins

    SciTech Connect

    Alexov, Emil; Mehler, Ernest L.; Baker, Nathan A.; Baptista, Antonio; Huang, Yong; Milletti, Francesca; Nielsen, Jens E.; Farrell, Damien; Carstensen, Tommy; Olsson, Mats H.; Shen, Jana K.; Warwicker, Jim; Williams, Sarah; Word, J Michael

    2011-12-15

    The pKa-cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pKa values and protein electrostatics in general. The first round of the pKa -cooperative, which challenged computational labs to carry out blind predictions against pKas experimentally determined in the laboratory of Bertrand Garcia-Moreno, was completed and results discussed at the Telluride meeting (July 6-10, 2009). This paper serves as an introduction to the reports submitted by the blind prediction participants that will be published in a special issue of PROTEINS: Structure, Function and Bioinformatics. Here we briefly outline existing approaches for pKa calculations, emphasizing methods that were used by the participants in calculating the blind pKa values in the first round of the cooperative. We then point out some of the difficulties encountered by the participating groups in making their blind predictions, and finally try to provide some insights for future developments aimed at improving the accuracy of pKa calculations.

  13. Effect of hydrogen bonds on pKa values: importance of networking.

    PubMed

    Shokri, Alireza; Abedin, Azardokht; Fattahi, Alireza; Kass, Steven R

    2012-06-27

    The pK(a) of an acyclic aliphatic heptaol ((HOCH(2)CH(2)CH(OH)CH(2))(3)COH) was measured in DMSO, and its gas-phase acidity is reported as well. This tertiary alcohol was found to be 10(21) times more acidic than tert-butyl alcohol in DMSO and an order of magnitude more acidic than acetic acid (i.e., pK(a) = 11.4 vs 12.3). This can be attributed to a 21.9 kcal mol(-1) stabilization of the charged oxygen center in the conjugate base by three hydrogen bonds and another 6.3 kcal mol(-1) stabilization resulting from an additional three hydrogen bonds between the uncharged primary and secondary hydroxyl groups. Charge delocalization by both the first and second solvation shells may be used to facilitate enzymatic reactions. Acidity constants of a series of polyols were also computed, and the combination of hydrogen-bonding and electron-withdrawing substituents was found to afford acids that are predicted to be extremely acidic in DMSO (i.e., pK(a) < 0). These hydrogen bond enhanced acids represent an attractive class of Brønsted acid catalysts.

  14. The IUPAC aqueous and non-aqueous experimental pKa data repositories of organic acids and bases.

    PubMed

    Slater, Anthony Michael

    2014-10-01

    Accurate and well-curated experimental pKa data of organic acids and bases in both aqueous and non-aqueous media are invaluable in many areas of chemical research, including pharmaceutical, agrochemical, specialty chemical and property prediction research. In pharmaceutical research, pKa data are relevant in ligand design, protein binding, absorption, distribution, metabolism, elimination as well as solubility and dissolution rate. The pKa data compilations of the International Union of Pure and Applied Chemistry, originally in book form, have been carefully converted into computer-readable form, with value being added in the process, in the form of ionisation assignments and tautomer enumeration. These compilations offer a broad range of chemistry in both aqueous and non-aqueous media and the experimental conditions and original reference for all pKa determinations are supplied. The statistics for these compilations are presented and the utility of the computer-readable form of these compilations is examined in comparison to other pKa compilations. Finally, information is provided about how to access these databases.

  15. Rational design of ratiometric near-infrared fluorescent pH probes with various pKa values, based on aminocyanine.

    PubMed

    Myochin, Takuya; Kiyose, Kazuki; Hanaoka, Kenjiro; Kojima, Hirotatsu; Terai, Takuya; Nagano, Tetsuo

    2011-03-16

    Novel ratiometric, near-infrared fluorescent pH probes with various pK(a) values have been designed and synthesized on the basis of aminocyanine bearing a diamine moiety, and their photochemical properties were evaluated. Under acidic conditions, these pH probes showed a 46- to 83-nm red shift of the absorption maximum. This change is sufficiently large to permit their use as ratiometric pH probes, and is reversible, whereas monoamine-substituted aminocyanines showed irreversible changes because of their instability under acidic conditions. Furthermore, the pK(a) values of these probes can be predicted from the calculated pK(a) values of the diamine moieties, obtained from the SciFinder database. This design strategy is very simple and flexible, and should be applicable to develop NIR pH probes for various applications.

  16. Revalidation and rationale for high pKa values of unconjugated bilirubin

    PubMed Central

    Ostrow, J Donald; Mukerjee, Pasupati

    2007-01-01

    Background Our prior solvent partition analysis, published in 1992, yielded pKa values for unconjugated bilirubin of about 8.1 and 8.4, but these results have been challenged and studies by other methods have suggested pKa values below 5.0. Methods We repeated our published solvent partition studies, using 14C-unconjugated bilirubin highly purified by extraction of residual labeled impurities from CHCl3 into an aqueous buffer, pH 7.0. Partition ratios at six pH values from 5.0 to 9.0 were determined by radioassay and compared with our prior values obtained by diazo assay. Results At pH values ranging from 4.8 to 9.2, stable aqueous/chloroform 14C-partition ratios did not differ significantly from our published partition ratios based on diazo assay. Conclusion These results support the high pKa values of unconjugated bilirubin, above 8.0, derived from our earlier solvent partition study. In both studies, our measurements were based on the rapid analysis of clearly under-saturated solutions of highly-purified bilirubin over a wide pH range, using properly purified and preserved solvents. No previous direct estimate of the aqueous pKa values of unconjugated bilirubin meets all these preconditions. Three theoretical factors acting in combination, each related to the unique, extensive internal H-bonding of the -COOH groups, are proposed to support high pKa values of unconjugated bilirubin in water: a) donation of an H-bond from the -OH moiety of the -COOH group, which is broken on ionization; b) hindered solvation of the -COO- group after ionization; and c) restricted rotation of the -COO- and -COOH groups. Our findings and rationale rebut methodological and theoretical criticisms leveled against our prior work. High pKa values for unconjugated bilirubin dictate that: a) bilirubin diacid, which readily diffuses across membranes and can cause neurotoxicity, is the dominant unbound bilirubin species of unconjugated bilirubin in plasma at physiological pH; b) at the near

  17. Determination of pK(a) Values of Hydrophobic Colorimetric pH Sensitive Probes in Nanospheres.

    PubMed

    Xie, Xiaojiang; Zhai, Jingying; Jarolímová, Zdeňka; Bakker, Eric

    2016-03-15

    A simple and novel method is proposed here for the first time to determine pK(a) values of chromogenic hydrophobic pH sensitive probes directly in nanospheres. pK(a) values can be obtained by measuring the pH response of the nanospheres (containing the probes and ion exchanger) followed by measuring the pH and Na(+) responses of the nanospheres (containing solvatochromic dyes and ion exchanger). The pK(a) values of four chromoionophores were successfully determined. This method is in principle also applicable to characterize colorimetric probes in other water immiscible nanomaterials.

  18. Prediction of the dissociation constant pKa of organic acids from local molecular parameters of their electronic ground state.

    PubMed

    Yu, Haiying; Kühne, Ralph; Ebert, Ralf-Uwe; Schüürmann, Gerrit

    2011-09-26

    A quantum chemical method has been developed to estimate the dissociation constant pK(a) of organic acids from their neutral molecular structures by employing electronic structure properties. The data set covers 219 phenols (including 29 phenols with intramolecular H-bonding), 150 aromatic carboxylic acids, 190 aliphatic carboxylic acids, and 138 alcohols, with pK(a) varying by 16 units (0.38-16.80). Optimized ground-state geometries employing the semiempirical AM1 Hamiltonian have been used to quantify the site-specific molecular readiness to donate or accept electron charge in terms of both charge-associated energies and energy-associated charges, augmented by an ortho substitution indicator for aromatic compounds. The resultant regression models yield squared correlation coefficients (r(2)) from 0.82 to 0.90 and root-mean-square errors (rms) from 0.39 to 0.70 pK(a) units, corresponding to an overall (subset-weighted) r(2) of 0.86. Simulated external validation, leave-10%-out cross-validation and target value scrambling demonstrate the statistical robustness and prediction power of the derived model suite. The low intercorrelation with prediction errors from the commercial ACD package provides opportunity for a consensus model approach, offering a pragmatic way for further increasing the confidence in prediction significantly. Interestingly, inclusion of calculated free energies of aqueous solvation does not improve the prediction performance, probably because of the limited precision provided by available continuum-solvation models.

  19. Computing pKa Values with a Mixing Hamiltonian Quantum Mechanical/Molecular Mechanical Approach.

    PubMed

    Liu, Yang; Fan, Xiaoli; Jin, Yingdi; Hu, Xiangqian; Hu, Hao

    2013-09-10

    Accurate computation of the pKa value of a compound in solution is important but challenging. Here, a new mixing quantum mechanical/molecular mechanical (QM/MM) Hamiltonian method is developed to simulate the free-energy change associated with the protonation/deprotonation processes in solution. The mixing Hamiltonian method is designed for efficient quantum mechanical free-energy simulations by alchemically varying the nuclear potential, i.e., the nuclear charge of the transforming nucleus. In pKa calculation, the charge on the proton is varied in fraction between 0 and 1, corresponding to the fully deprotonated and protonated states, respectively. Inspired by the mixing potential QM/MM free energy simulation method developed previously [H. Hu and W. T. Yang, J. Chem. Phys. 2005, 123, 041102], this method succeeds many advantages of a large class of λ-coupled free-energy simulation methods and the linear combination of atomic potential approach. Theory and technique details of this method, along with the calculation results of the pKa of methanol and methanethiol molecules in aqueous solution, are reported. The results show satisfactory agreement with the experimental data.

  20. Determination of pKa values of some novel benzimidazole salts by using a new approach with (1)H NMR spectroscopy.

    PubMed

    Mumcu, Akın; Küçükbay, Hasan

    2015-12-01

    Benzimidazoles and their derivatives including imidazole are studied widely because they exist in the structure of natural products and different drugs. pKa values are extremely important for drug discovery and improvement in order to determine pharmacokinetic and pharmacodynamic features such as permeation through biological barriers, interactions with the target area or side effects. Acid-base features (pKa ) have great importance not only for physiological characteristics but also for being used as a ligand or changing physico-chemical features by turning benzimidazoles into salts. Within the scope of this study, a variety of new benzimidazole salts were synthesized, and their characterizations were made by NMR spectroscopy, FTIR spectroscopy and element analysis techniques. The pKa values of synthesized benzimidazole salts were determined by inflection point approach using integration values obtained with (1) H NMR spectroscopy and Henderson-Hasselbalch analysis. pKa values of some benzimidazole salts were also determined by potentiometric methods in order to compare those of NMR spectroscopy results.

  1. Determination of pKa values of some novel benzimidazole salts by using a new approach with (1)H NMR spectroscopy.

    PubMed

    Mumcu, Akın; Küçükbay, Hasan

    2015-12-01

    Benzimidazoles and their derivatives including imidazole are studied widely because they exist in the structure of natural products and different drugs. pKa values are extremely important for drug discovery and improvement in order to determine pharmacokinetic and pharmacodynamic features such as permeation through biological barriers, interactions with the target area or side effects. Acid-base features (pKa ) have great importance not only for physiological characteristics but also for being used as a ligand or changing physico-chemical features by turning benzimidazoles into salts. Within the scope of this study, a variety of new benzimidazole salts were synthesized, and their characterizations were made by NMR spectroscopy, FTIR spectroscopy and element analysis techniques. The pKa values of synthesized benzimidazole salts were determined by inflection point approach using integration values obtained with (1) H NMR spectroscopy and Henderson-Hasselbalch analysis. pKa values of some benzimidazole salts were also determined by potentiometric methods in order to compare those of NMR spectroscopy results. PMID:26256272

  2. pK(a) values of the monohydroxylated polychlorinated biphenyls (OH-PCBs), polybrominated biphenyls (OH-PBBs), polychlorinated diphenyl ethers (OH-PCDEs), and polybrominated diphenyl ethers (OH-PBDEs).

    PubMed

    Rayne, Sierra; Forest, Kaya

    2010-09-01

    The SPARC software program aqueous pK(a) prediction module was validated against corresponding experimental acidity constants for chlorinated and brominated phenols and the limited experimental aqueous pK(a) data sets for monohydroxylated polychlorinated biphenyls (OH-PCBs), polychlorinated diphenyl ethers (OH-PCDEs), and polybrominated diphenyl ethers (OH-PBDEs). pK(a) values were then estimated for all 837 monohydroxylated mono- through nona-halogenated congeners in each of the OH-PCB, OH-PCDE, and OH-PBDE classes, as well as for the monohydroxylated polybrominated biphenyls (OH-PBBs), giving a total of 3348 compounds. Large intrahomolog pK(a) variation by up to six units is expected within each contaminant class, with pK(a) values ranging from about 4 to 11 dependent on the degree and pattern of halogenation. Increasing halogenation generally decreased the average pK(a) within each homolog group. Significant intrahomolog differences in pK(a) values exist between OH-PCB, OH-PBB, OH-PCDE, and OH-PBDE congeners, including large acidity constant variation between isomers with equivalent halogenation patterns but varying location of the hydroxy moiety. Congener specific pH dependent investigations into the partitioning and degradation behaviors of these compounds are necessary, including greater consideration of analyte ionization effects during their extraction and analysis. PMID:20658412

  3. Predicting pK(a) values of substituted phenols from atomic charges: comparison of different quantum mechanical methods and charge distribution schemes.

    PubMed

    Svobodová Vareková, Radka; Geidl, Stanislav; Ionescu, Crina-Maria; Skrehota, Ondrej; Kudera, Michal; Sehnal, David; Bouchal, Tomás; Abagyan, Ruben; Huber, Heinrich J; Koca, Jaroslav

    2011-08-22

    The acid dissociation (ionization) constant pK(a) is one of the fundamental properties of organic molecules. We have evaluated different computational strategies and models to predict the pK(a) values of substituted phenols using partial atomic charges. Partial atomic charges for 124 phenol molecules were calculated using 83 approaches containing seven theory levels (MP2, HF, B3LYP, BLYP, BP86, AM1, and PM3), three basis sets (6-31G*, 6-311G, STO-3G), and five population analyses (MPA, NPA, Hirshfeld, MK, and Löwdin). The correlations between pK(a) and various atomic charge descriptors were examined, and the best descriptors were selected for preparing the quantitative structure-property relationship (QSPR) models. One QSPR model was created for each of the 83 approaches to charge calculation, and then the accuracy of all these models was analyzed and compared. The pK(a)s predicted by most of the models correlate strongly with experimental pK(a) values. For example, more than 25% of the models have correlation coefficients (R²) greater than 0.95 and root-mean-square errors smaller than 0.49. All seven examined theory levels are applicable for pK(a) prediction from charges. The best results were obtained for the MP2 and HF level of theory. The most suitable basis set was found to be 6-31G*. The 6-311G basis set provided slightly weaker correlations, and unexpectedly also, the STO-3G basis set is applicable for the QSPR modeling of pK(a). The Mulliken, natural, and Löwdin population analyses provide accurate models for all tested theory levels and basis sets. The results provided by the Hirshfeld population analysis were also acceptable, but the QSPR models based on MK charges show only weak correlations.

  4. NMR determination of lysine pKa values in the Pol lambda lyase domain: mechanistic implications.

    PubMed

    Gao, Guanghua; DeRose, Eugene F; Kirby, Thomas W; London, Robert E

    2006-02-14

    The base excision repair (BER) process requires removal of an abasic deoxyribose-5-phosphate group, a catalytic activity that has been demonstrated for the N-terminal 8 kDa domain of DNA polymerase beta (Pol beta), and for the homologous domain of DNA polymerase lambda (Pol lambda). Previous studies have demonstrated that this activity results from formation of a Schiff base adduct of the abasic deoxyribose C-1' with a lysine residue (K312 in the case of Pol lambda), followed by a beta-elimination reaction. To better understand the underlying chemistry, we have determined pKa values for the lysine residues in the Pol lambda lyase domain labeled with [epsilon-13C]lysine. At neutral pH, the H(epsilon) protons on 3 of the 10 lysine residues in this domain, K287, K291, and K312, exhibit chemical shift inequivalence that results from immobilization of the lysyl side chains. For K287 and K291, this results from the K287-E261 and K291-E298 salt bridge interactions, while for K312, immobilization apparently results from steric and hydrogen-bonding interactions that constrain the position of the lysine side chain. The pKa value of K312 is depressed to 9.58, a value indicating that at physiological pH K312 will exist predominantly in the protonated form. Titration of the domain with hairpin DNA containing a 5'-tetrahydrofuran terminus to model the abasic site produced shifts of the labeled lysine resonances that were in fast exchange but appeared to be complete at a stoichiometry of approximately 1:1.3, consistent with a dissociation constant of approximately 1 microM. The epsilon-proton shifts of K273 were the most sensitive to the addition of the DNA, apparently due to changes in the relative orientation between K273 and W274 in the DNA complex. The average pKa values increased by 0.55, consistent with the formation of some DNA-lysine salt bridges and with the general pH increase expected to result from a reduction in the net positive charge of the complex. A general

  5. pH-dependent pKa values in proteins--a theoretical analysis of protonation energies with practical consequences for enzymatic reactions.

    PubMed

    Bombarda, Elisa; Ullmann, G Matthias

    2010-02-11

    Because of their central importance for understanding enzymatic mechanisms, pK(a) values are of great interest in biochemical research. It is common practice to determine pK(a) values of amino acid residues in proteins from NMR or FTIR titration curves by determining the pH at which the protonation probability is 50%. The pH dependence of the free energy required to protonate this residue is then determined from the linear relationship DeltaG(prot) = RT ln 10 (pH-pK(a)), where R is the gas constant and T the absolute temperature. However, this approach neglects that there can be important electrostatic interactions in the proteins that can shift the protonation energy. Even if the titration curves seem to have a standard sigmoidal shape, the protonation energy of an individual site in a protein may depend nonlinearly on pH. To account for this nonlinear dependence, we show that it is required to introduce pK(a) values for individual sites in proteins that depend on pH. Two different definitions are discussed. One definition is based on a rearranged Henderson-Hasselbalch equation, and the other definition is based on an equation that was used by Tanford and Roxby to approximate titration curves of proteins. In the limiting case of weak interactions, the two definitions lead to nearly the same pK(a) value. We discuss how these two differently defined pK(a) values are related to the free energy change required to protonate a site. Using individual site pK(a) values, we demonstrate on simple model systems that the interactions between protonatable residues in proteins can help to maintain the energy required to protonate a site in the protein nearly constant over a wide pH range. We show with the example of RNase T1 that such a mechanism to keep the protonation energy constant is used in enzymes. The pH dependence of pK(a) values may be an important concept in enzyme catalysis. Neglecting this concept, important features of enzymes may be missed, and the enzymatic

  6. Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis.

    PubMed Central

    Liu, T.; Ryan, M.; Dahlquist, F. W.; Griffith, O. H.

    1997-01-01

    Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa. PMID:9300493

  7. Determination of pKa for Dithiophosphinic Acids using Density Functional Theory

    SciTech Connect

    Michael T. Benson; Megan L. Moser; Dean R. Peterman; Adriana Dinescu

    2008-10-01

    Symmetric aromatic dithiophosphinic acids of the form (XC6H4)2P(=S)(SH), where X = H, o-CH3, p-CH3, p-Cl, p-F, o-CF3, m-CF3, and p-CF3, and asymmetric aromatic acids of the form (X’C6H4)(X”C6H4)P(=S)(SH), where X’ = o-CF3, X” = m-CF3; X’ = H, X” = o-CF3, have been investigated using B3LYP/Gaussian03. Solvation was included in the calculations using the CPCM continuum solvation method. Using the data from vibrational frequency calculations, the pKa was calculated for the acids, and compared to Cyanex-301. The unexpectedly high pKa for bis(o-trifluoromethylphenyl)dithiophosphinic acid, when compared to the ortho-meta, meta-meta, and para-para isomers, is rationalized by e- repulsion between nearby fluorines and the sulfurs in the anion. This repulsion destabilizes the anion, versus the other isomers, thus raising the pKa.

  8. Evaluation of Thiol Raman Activities and pKa Values Using Internally Referenced Raman-Based pH Titration.

    PubMed

    Suwandaratne, Nuwanthi; Hu, Juan; Siriwardana, Kumudu; Gadogbe, Manuel; Zhang, Dongmao

    2016-04-01

    Thiols, including organothiol and thiol-containing biomolecules, are among the most important classes of chemicals that are used broadly in organic synthesis, biological chemistry, and nanosciences. Thiol pKa values are key indicators of thiol reactivity and functionality. Reported herein is an internally referenced Raman-based pH titration method that enables reliable quantification of thiol pKa values for both mono- and dithiols in water. The degree of thiol ionization is monitored directly using the peak intensity of the S-H stretching feature in the 2600 cm(-1) region relative to an internal reference peak as a function of the titration solution's pH. The thiol pKa values and Raman activity relative to its internal reference were then determined by curve fitting the experimental data with equations derived on the basis of the Henderson-Hasselbalch equation. Using this Raman titration method, we determined for the first time the first and second thiol pKa values for 1,2-benzenedithiol in water. This Raman-based method is convenient to implement, and its underlying theory is easy to follow. It should therefore have broad application for thiol pKa determinations and verification.

  9. Evaluation of thiol Raman activities and pKa values using internally referenced Ramanbased pH titration

    NASA Astrophysics Data System (ADS)

    Suwandaratne, Nuwanthi

    Thiols are one of the most important classes of chemicals used broadly in organic synthesis, biological chemistry, and nanosciences. Thiol pKa values are key indicators of thiol reactivity and functionality. This study is an internally-referenced Raman-based pH titration method that enables reliable quantification of thiol pKa values for both mono- and di-thiols in water. The degree of thiol ionization is monitored directly using the peak intensity of the S-H stretching feature relative to an internal reference peak as a function of solution pH. The thiol pKa values and Raman activity relative to its internal reference were then determined by curve-fitting the experimental data with equations derived on the basis of the Henderson-Hasselbalch equation. Using this Raman titration method, first and second thiol pKa values for 1,2-benzenedithol in water were determined for the first time. This method is convenient to implement and its underlying theory is easy to follow.

  10. Ab Initio MD Simulations of the Brønsted Acidity of Glutathione in Aqueous Solutions: Predicting pKa Shifts of the Cysteine Residue.

    PubMed

    Tummanapelli, Anil Kumar; Vasudevan, Sukumaran

    2015-12-10

    The tripeptide glutathione (GSH) is one of the most abundant peptides and the major repository for nonprotein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thiol-disulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influence the redox couple, and hence the pKa value of the cysteine residue of GSH is critical to its functioning. Here we report ab initio Car-Parrinello molecular dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. We show that the free-energy landscape for the protonation-deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared with the isolated cysteine amino acid.

  11. Comprehensive determination of protein tyrosine pKa values for photoactive yellow protein using indirect 13C NMR spectroscopy.

    PubMed

    Oktaviani, Nur Alia; Pool, Trijntje J; Kamikubo, Hironari; Slager, Jelle; Scheek, Ruud M; Kataoka, Mikio; Mulder, Frans A A

    2012-02-01

    Upon blue-light irradiation, the bacterium Halorhodospira halophila is able to modulate the activity of its flagellar motor and thereby evade potentially harmful UV radiation. The 14 kDa soluble cytosolic photoactive yellow protein (PYP) is believed to be the primary mediator of this photophobic response, and yields a UV/Vis absorption spectrum that closely matches the bacterium's motility spectrum. In the electronic ground state, the para-coumaric acid (pCA) chromophore of PYP is negatively charged and forms two short hydrogen bonds to the side chains of Glu-46 and Tyr-42. The resulting acid triad is central to the marked pH dependence of the optical-absorption relaxation kinetics of PYP. Here, we describe an NMR approach to sequence-specifically follow all tyrosine side-chain protonation states in PYP from pH 3.41 to 11.24. The indirect observation of the nonprotonated (13)C(γ) resonances in sensitive and well-resolved two-dimensional (13)C-(1)H spectra proved to be pivotal in this effort, as observation of other ring-system resonances was hampered by spectral congestion and line-broadening due to ring flips. We observe three classes of tyrosine residues in PYP that exhibit very different pK(a) values depending on whether the phenolic side chain is solvent-exposed, buried, or hydrogen-bonded. In particular, our data show that Tyr-42 remains fully protonated in the pH range of 3.41-11.24, and that pH-induced changes observed in the photocycle kinetics of PYP cannot be caused by changes in the charge state of Tyr-42. It is therefore very unlikely that the pCA chromophore undergoes changes in its electrostatic interactions in the electronic ground state. PMID:22325281

  12. The intrinsic pKa values for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in monolayers deposited on mercury electrodes.

    PubMed Central

    Moncelli, M R; Becucci, L; Guidelli, R

    1994-01-01

    The intrinsic pKa values of the phosphate groups of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and of the phosphate and carboxyl groups of phosphatidylserine (PS) in self-organized monolayers deposited on a hanging mercury drop electrode were determined by a novel procedure based on measurements of the differential capacity C of this lipid-coated electrode. In view of the Gouy-Chapman theory, plots of 1/C at constant bulk pH and variable KCl concentration against the reciprocal of the calculated diffuse-layer capacity Cd,0 at zero charge exhibit slopes that decrease from an almost unit value to vanishingly low values as the absolute value of the charge density on the lipid increases from zero to approximately 2 microC cm-2. The intrinsic pKa values so determined are 0.5 for PE and 0.8 for PC. The plots of 1/C against 1/Cd,0 for pure PS exhibit slopes that pass from zero to a maximum value and then back to zero as pH is varied from 7.5 to 3, indicating that the charge density of the lipid film passes from slight negative to slight positive values over this pH range. An explanation for this anomalous behavior, which is ascribed to the phosphate group of PS, is provided. Interdispersion of PS and PC molecules in the film decreases the "formal" pKa value of the latter group by about three orders of magnitude. PMID:8075331

  13. Quantum chemical investigation on indole: vibrational force field and theoretical determination of its aqueous pK(a) value.

    PubMed

    Pietropolli Charmet, Andrea; Quartarone, Giuseppe; Ronchin, Lucio; Tortato, Claudio; Vavasori, Andrea

    2013-08-01

    Indole and its derivatives are molecules which play important roles in different fields, from biology to pharmacology. Here we report a thorough investigation on the anharmonic force fields of indole as well as the ab initio determinations of its gas phase basicity and aqueous pK(a) value. For the geometry optimizations, the calculations have been performed using both density functional (DFT) and second-order Møller-Plesset (MP2) levels of theory employing different basis sets. Anharmonic force fields have been obtained employing both the B3LYP and the B97-1 functionals and an hybrid approach: the best agreement to the experimental data has been determined employing the B3LYP functional combined with the recently developed N07D basis set (mean unsigned error, MUE, of 5.1 cm(-1) and a root-mean-square error, RMSE, of 7.2 cm(-1)). Gas phase basicity and proton affinity have been computed employing several computational schemes, namely the G3 and G4 Gaussian models, the complete basis set (CBS) extrapolation methods of Petersson and co-workers, several DFT calculations, and different hybrid extrapolation schemes based on combining single-point energy calculations performed at MP2 as well as at coupled cluster level of theory with single, double and perturbative triple excitations, CCSD(T). Regarding the aqueous pK(a) computations, two implicit solvation models (SMD and SM8) have been employed to determine the free energy of solvation and the corresponding pKa value.

  14. Determination of pKa and Hydration Constants for a Series of α-Keto-Carboxylic Acids Using Nuclear Magnetic Resonance Spectrometry.

    PubMed

    Lopalco, Antonio; Douglas, Justin; Denora, Nunzio; Stella, Valentino J

    2016-02-01

    The determination of the acid-base dissociation constants, and thus the pKa values, of α-keto acids such as pyruvic acid is complex because of the existence of these acids in their hydrated and nonhydrated or oxo state. Equilibria involved in the hydration and dehydration of the α-keto group of pyruvic acid and three other α-keto acids, 3-methyl-2-oxobutanoic acid, 4-methyl-2-oxopentanoic acid, and 2-oxo-2-phenylacetic acid, were investigated by proton and carbon nuclear magnetic resonance spectrometry, at constant ionic strength, 0.15, and 25 °C. Dissociation constants for the oxo (pKa(oxo)) and hydrated (pKa(hyd)) acids of each compound were estimated from the change in the degree of hydration with changes in pH and directly from the changes in chemical shifts of various hydrogen and carbons nuclei with pH. α-Keto acids showed greater hydration in their acidic forms than their carboxylate forms. The degree of hydration was sensitive to steric and electronic/resonance factors. As expected, the oxo forms of the acids were stronger acids compared with their hydrated analogs, and their dissociation constants were also sensitive to steric and electronic factors.

  15. Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways.

    PubMed

    Yu, Zi-Li; Li, Dian-Qi; Huang, Xiang-Yu; Xing, Xin; Yu, Ru-Qing; Li, Zhi; Li, Zu-Bing

    2016-02-01

    Lysophosphatidic acid (LPA) is an efficient, bioactive phospholipid involved in various biological processes. In this study, LPA-induced connective tissue growth factor (CTGF/CCN2) expression and the underlying mechanisms were investigated using the MC3T3-E1 cell line. The MC3T3-E1 cells were stimulated with an inhibitor of LPA receptors, an activator and inhibitor of protein kinase C (PKC) and protein kinase A (PKA) for indicated periods of time. RT-qPCR and western blot analyses were used to measure the expression levels of CCN2. Immunofluorescence staining was used to observe the translocation of PKC. The mRNA expression level of CCN2 was increased following stimulation of the cells with LPA; LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation with LPA. This increase was accompanied by CCN2 protein synthesis. LPA receptor1/3 was inhibited by Ki16425, a specific inhibitor of LPA1/3; as a result, the LPA-induced increase in CCN2 expression was abrogated. LPA also induced the membrane translocation of PKC and enhanced PKC activity in the osteoblasts. Pre-treatment of the osteoblasts with staurosporine prevented the increase in CCN2 expression by induced by LPA, and the activation of PKC by phorbol 12-myristate 13-acetate (PMA) enhanced CCN2 expression, indicating that the PKC pathway is involved in the LPA-induced increase in CCN2 expression. The interference of PKA signaling also led to the induction of CCN2 expresion by LPA. These data indicate that LPA increases CCN2 expression through the activation of PKC and PKA. Thus, the regulatory functions of the PKC and PKA pathways are implicated in the LPA-induced increase in CCN2 expression.

  16. Theoretical calculations of stability constants and pKa values of metal complexes in solution: application to pyridoxamine-copper(II) complexes and their biological implications in AGE inhibition.

    PubMed

    Casasnovas, Rodrigo; Ortega-Castro, Joaquín; Donoso, Josefa; Frau, Juan; Muñoz, Francisco

    2013-10-14

    Accurate prediction of thermodynamic constants of chemical reactions in solution is one of the current challenges in computational chemistry. We report a scheme for predicting stability constants (log β) and pKa values of metal complexes in solution by means of calculating free energies of ligand- and proton-exchange reactions using Density Functional Theory calculations in combination with a continuum solvent model. The accuracy of the predicted log β and pKa values (mean absolute deviations of 1.4 and 0.2 units respectively) is equivalent to the experimental uncertainties. This theoretical methodology provides direct knowledge of log β and pKa values of major and minor species, so it is of potential use in combination with experimental techniques to obtain a detailed description of the microscopic equilibria. In particular, the proposed methodology is shown to be especially useful for obtaining the real acidity constants of those chelates where the metal-ligand coordination changes as a result of ligand deprotonation. The stability and acidity constants of pyridoxamine-Cu(2+) chelates calculated with the proposed methodology show that pyridoxamine is an efficient scavenging agent of Cu(2+) under physiological pH conditions. This is of special interest as Cu(2+) overload is involved in the formation of advanced glycation end-products (AGEs) and their associated degenerative medical conditions. PMID:23999915

  17. Linear free-energy relationships between a single gas-phase ab initio equilibrium bond length and experimental pKa values in aqueous solution.

    PubMed

    Alkorta, Ibon; Popelier, Paul L A

    2015-02-01

    Remarkably simple yet effective linear free energy relationships were discovered between a single ab initio computed bond length in the gas phase and experimental pKa values in aqueous solution. The formation of these relationships is driven by chemical features such as functional groups, meta/para substitution and tautomerism. The high structural content of the ab initio bond length makes a given data set essentially divide itself into high correlation subsets (HCSs). Surprisingly, all molecules in a given high correlation subset share the same conformation in the gas phase. Here we show that accurate pKa values can be predicted from such HCSs. This is achieved within an accuracy of 0.2 pKa units for 5 drug molecules.

  18. The effects of solvent polarity and pKa on the absorption of solvents into poly(glutaric acid-glycerol) films

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, solvent absorption into the matrices of poly(glutaric acid-glycerol) films has been evaluated. It was determined that the combined effects of polarity and the size and shape of the solvent molecule, rather than pKa, have the most significant influence on absorption into the films. P...

  19. Stacking and determination of phenazine-1-carboxylic acid with low pKa in soil via moving reaction boundary formed by alkaline and double acidic buffers in capillary electrophoresis.

    PubMed

    Sun, Chong; Yang, Xiao-Di; Fan, Liu-Yin; Zhang, Wei; Xu, Yu-Quan; Cao, Cheng-Xi

    2011-04-01

    As shown herein, a normal moving reaction boundary (MRB) formed by an alkaline buffer and a single acidic buffer had poor stacking to the new important plant growth promoter of phenazine-1-carboxylic acid (PCA) in soil due to the leak induced by its low pK(a). To stack the PCA with low pK(a) efficiently, a novel stacking system of MRB was developed, which was formed by an alkaline buffer and double acidic buffers (viz., acidic sample and blank buffers). With the novel system, the PCA leaking into the blank buffer from the sample buffer could be well stacked by the prolonged MRB formed between the alkaline buffer and blank buffer. The relevant mechanism of stacking was discussed briefly. The stacking system, coupled with sample pretreatment, could achieve a 214-fold increase of PCA sensitivity under the optimal conditions (15 mM (pH 11.5) Gly-NaOH as the alkaline buffer, 15 mM (pH 3.0) Gly-HCl-acetonitrile (20%, v/v) as the acidic sample buffer, 15 mM (pH 3.0) Gly-HCl as the blank buffer, 3 min 13 mbar injection of double acidic buffers, benzoic acid as the internal standard, 75 μm i.d. × 53 cm (44 cm effective length) capillary, 25 kV and 248 nm). The limit of detection of PCA in soil was decreased to 17 ng/g, the intra-day and inter-day precision values (expressed as relative standard deviations) were 3.17-4.24% and 4.17-4.87%, respectively, and the recoveries of PCA at three concentration levels changed from 52.20% to 102.61%. The developed method could be used for the detection of PCA in soil at trace level.

  20. Experimental approaches for measuring pKa's in RNA and DNA.

    PubMed

    Thaplyal, Pallavi; Bevilacqua, Philip C

    2014-01-01

    RNA and DNA carry out diverse functions in biology including catalysis, splicing, gene regulation, and storage of genetic information. Interest has grown in understanding how nucleic acids perform such sophisticated functions given their limited molecular repertoire. RNA can fold into diverse shapes that often perturb pKa values and allow it to ionize appreciably under biological conditions, thereby extending its molecular diversity. The goal of this chapter is to enable experimental measurement of pKa's in RNA and DNA. A number of experimental methods for measuring pKa values in RNA and DNA have been developed over the last 10 years, including RNA cleavage kinetics; UV-, fluorescence-, and NMR-detected pH titrations; and Raman crystallography. We begin with general considerations for choosing a pKa assay and then describe experimental conditions, advantages, and disadvantages for these assays. Potential pitfalls in measuring a pKa are provided including the presence of apparent pKa's due to a kinetic pKa or coupled acid- and alkali-promoted RNA unfolding, as well as degradation of RNA, precipitation of metal hydroxides and poor baselines. Use of multiple data fitting procedures and the study of appropriate mutants are described as ways to avoid some of these pitfalls. Application of these experimental methods to RNA and DNA will increase the number of available nucleic acid pKa values in the literature, which should deepen insight into biology and provide benchmarks for pKa calculations. Future directions for measuring pKa's in nucleic acids are discussed.

  1. Novel zinc(II)phthalocyanines bearing azo-containing schiff base: Determination of pKa values, absorption, emission, enzyme inhibition and photochemical properties

    NASA Astrophysics Data System (ADS)

    Kantar, Cihan; Mavi, Vildan; Baltaş, Nimet; İslamoğlu, Fatih; Şaşmaz, Selami

    2016-10-01

    Azo-containing schiff bases are well known and there are many studies about their various properties in literature. However, phthalocyanines bearing azo-containing schiff bases, their spectral, analytical and biological properties are unknown. Therefore, new zinc (II) phthalocyanines bearing azo-containing schiff base were synthesized and investigated to determine pKa values, absorption, emission, enzyme inhibition and photochemical properties. Emission spectra were reported and large Stokes shift values were determined for all compounds, indicating that all molecules exhibit excited state intramolecular proton transfer. These phthalocyanines were the first examples of phthalocyanine showing excited state intramolecular proton transfer. Singlet oxygen quantum yields of zinc (II) phthalocyanines were determined. pKa values and indicator properties of all compounds were investigated by potentiometry. All compounds were assayed for inhibitory activity against bovine milk xanthine oxidase and acetylcholinesterase enzyme in vitro. Compound 2 showed the high inhibitory effect against xanthine oxidase (IC50 = 0.24 ± 0.01 μM). However, phthalocyanine compounds did not show enzyme inhibitor behavior.

  2. Comparative analysis of QSAR models for predicting pK(a) of organic oxygen acids and nitrogen bases from molecular structure.

    PubMed

    Yu, Haiying; Kühne, Ralph; Ebert, Ralf-Uwe; Schüürmann, Gerrit

    2010-11-22

    For 1143 organic compounds comprising 580 oxygen acids and 563 nitrogen bases that cover more than 17 orders of experimental pK(a) (from -5.00 to 12.23), the pK(a) prediction performances of ACD, SPARC, and two calibrations of a semiempirical quantum chemical (QC) AM1 approach have been analyzed. The overall root-mean-square errors (rms) for the acids are 0.41, 0.58 (0.42 without ortho-substituted phenols with intramolecular H-bonding), and 0.55 and for the bases are 0.65, 0.70, 1.17, and 1.27 for ACD, SPARC, and both QC methods, respectively. Method-specific performances are discussed in detail for six acid subsets (phenols and aromatic and aliphatic carboxylic acids with different substitution patterns) and nine base subsets (anilines, primary, secondary and tertiary amines, meta/para-substituted and ortho-substituted pyridines, pyrimidines, imidazoles, and quinolines). The results demonstrate an overall better performance for acids than for bases but also a substantial variation across subsets. For the overall best-performing ACD, rms ranges from 0.12 to 1.11 and 0.40 to 1.21 pK(a) units for the acid and base subsets, respectively. With regard to the squared correlation coefficient r², the results are 0.86 to 0.96 (acids) and 0.79 to 0.95 (bases) for ACD, 0.77 to 0.95 (acids) and 0.85 to 0.97 (bases) for SPARC, and 0.64 to 0.87 (acids) and 0.43 to 0.83 (bases) for the QC methods, respectively. Attention is paid to structural and method-specific causes for observed pitfalls. The significant subset dependence of the prediction performances suggests a consensus modeling approach. PMID:21033677

  3. The pKa Distribution of Drugs: Application to Drug Discovery

    PubMed Central

    Manallack, David T.

    2007-01-01

    The acid-base dissociation constant (pKa) of a drug is a key physicochemical parameter influencing many biopharmaceutical characteristics. While this has been well established, the overall proportion of non-ionizable and ionizable compounds for drug-like substances is not well known. Even less well known is the overall distribution of acid and base pKa values. The current study has reviewed the literature with regard to both the proportion of ionizable substances and pKa distributions. Further to this a set of 582 drugs with associated pKa data was thoroughly examined to provide a representative set of observations. This was further enhanced by delineating the compounds into CNS and non-CNS drugs to investigate where differences exist. Interestingly, the distribution of pKa values for single acids differed remarkably between CNS and non-CNS substances with only one CNS compound having an acid pKa below 6.1. The distribution of basic substances in the CNS set also showed a marked cut off with no compounds having a pKa above 10.5. The pKa distributions of drugs are influenced by two main drivers. The first is related to the nature and frequency of occurrence of the functional groups that are commonly observed in pharmaceuticals and the typical range of pKa values they span. The other factor concerns the biological targets these compounds are designed to hit. For example, many CNS targets are based on seven transmembrane G protein-coupled receptors (7TM GPCR) which have a key aspartic acid residue known to interact with most ligands. As a consequence, amines are mostly present in the ligands that target 7TM GPCR’s and this influences the pKa profile of drugs containing basic groups. For larger screening collections of compounds, synthetic chemistry and the working practices of the chemists themselves can influence the proportion of ionizable compounds and consequent pKa distributions. The findings from this study expand on current wisdom in pKa research and have

  4. Formation and stability of N-heterocyclic carbenes in water: the carbon acid pKa of imidazolium cations in aqueous solution.

    PubMed

    Amyes, Tina L; Diver, Steven T; Richard, John P; Rivas, Felix M; Toth, Krisztina

    2004-04-01

    We report second-order rate constants kDO (M-1 s-1) for exchange for deuterium of the C(2)-proton of a series of simple imidazolium cations to give the corresponding singlet imidazol-2-yl carbenes in D2O at 25 degrees C and I = 1.0 (KCl). Evidence is presented that the reverse protonation of imidazol-2-yl carbenes by solvent water is limited by solvent reorganization and occurs with a rate constant of kHOH = kreorg = 10(11) s-1. The data were used to calculate reliable carbon acid pK(a)s for ionization of imidazolium cations at C(2) to give the corresponding singlet imidazol-2-yl carbenes in water: pKa = 23.8 for the imidazolium cation, pKa = 23.0 for the 1,3-dimethylimidazolium cation, pKa = 21.6 for the 1,3-dimethylbenzimidazolium cation, and pKa = 21.2 for the 1,3-bis-((S)-1-phenylethyl)benzimidazolium cation. The data also provide the thermodynamic driving force for a 1,2-hydrogen shift at a singlet carbene: K12 = 5 x 10(16) for rearrangement of the parent imidazol-2-yl carbene to give neutral imidazole in water at 298 K, which corresponds to a favorable Gibbs free energy change of 23 kcal/mol. We present a simple rationale for the observed substituent effects on the thermodynamic stability of N-heterocyclic carbenes relative to a variety of neutral and cationic derivatives that emphasizes the importance of the choice of reference reaction when assessing the stability of N-heterocyclic carbenes.

  5. pKa determination by ¹H NMR spectroscopy - an old methodology revisited.

    PubMed

    Bezençon, Jacqueline; Wittwer, Matthias B; Cutting, Brian; Smieško, Martin; Wagner, Bjoern; Kansy, Manfred; Ernst, Beat

    2014-05-01

    pKa values of acids and protonated bases have an essential impact on organic synthesis, medicinal chemistry, and material and food sciences. In drug discovery and development, they are of utmost importance for the prediction of pharmacokinetic and pharmacodynamic properties. To date, various methods for the determination of pKa values are available, including UV-spectroscopic, potentiometric, and capillary electrophoretic techniques. An additional option is provided by nuclear magnetic resonance (NMR) spectroscopy. The underlying principle is the alteration of chemical shifts of NMR-active nuclei (e.g., (13)C and (1)H) depending on the protonation state of adjacent acidic or basic sites. When these chemical shifts are plotted against the pH, the inflection point of the resulting sigmoidal curve defines the pKa value. Although pKa determinations by (1)H NMR spectroscopy are reported for numerous cases, the potential of this approach is not yet fully evaluated. We therefore revisited this method with a diverse set of test compounds covering a broad range of pKa values (pKa 0.9-13.8) and made a comparison with four commonly used approaches. The methodology revealed excellent correlations (R(2)=0.99 and 0.97) with electropotentiometric and UV spectroscopic methods. Moreover, the comparison with in silico results (Epik and Marvin) also showed high correlations (R(2)=0.92 and 0.94), further confirming the reliability and utility of this approach. PMID:24462329

  6. Nuclear magnetic resonance studies on the pKa values and interaction of ionizable groups in bromelain inhibitor VI from pineapple stem.

    PubMed

    Hatano, Ken-ichi; Kojima, Masaki; Tanokura, Masaru; Takahashi, Kenji

    2003-01-01

    Bromelain inhibitor VI (BI-VI), a cysteine proteinase inhibitor from pineapple stem, is a unique double-chain molecule composed of two distinct domains A and B. In order to clarify the molecular mechanism of the proteinase-inhibitor interaction, we investigated the electrostatic properties of this inhibitor. The inhibitory activity toward bromelain was revealed to be maximal at pH 3-4 and the gross conformation to be stable over a wide range of pH. Based on these results, pH titration experiments were performed on the proton resonances of BI-VI in the pH range of 1.5-9.9, and pKa values (pKexp) were determined for all carboxyl groups and alpha-amino groups. The pKexp were also compared with theoretical values calculated from the NMR-derived structures of BI-VI. The electrostatic surface potential map constructed using the pKexp values revealed that BI-VI possesses continuous negatively charged and scattered positively charged regions on the molecular surface and both regions appear to serve for docking properly with a basic target enzyme. Furthermore, it was suggested that the ionic interaction of the inhibitor with the target enzyme is primarily important for the inhibition, which seems to involve some carboxyl groups in the inhibitor and a thiol group in the proteinase.

  7. Evaluation of Methods for the Calculation of the pKa of Cysteine Residues in Proteins.

    PubMed

    Awoonor-Williams, Ernest; Rowley, Christopher N

    2016-09-13

    Methods for the calculation of the pKa ionizable amino acids are valuable tools for understanding pH-dependent properties of proteins. Cysteine is unique among the amino acids because of the chemical reactivity of its thiol group (S-H), which plays an instrumental role in several biochemical and regulatory functions. The acidity of noncatalytic cysteine residues is a factor in their susceptibility to chemical modification. Despite the plethora of existing pKa computing methods, no definitive protocol exists for accurately calculating the pKa's of cysteine residues in proteins. A cysteine pKa test set was developed, which is comprised of 18 cysteine residues in 12 proteins where the pKa's have been determined experimentally and an experimental structure is available. The pKa's of these residues were calculated using three methods that use an implicit solvent model (H++, MCCE, and PROPKA) and an all-atom replica-exchange thermodynamic integration approach with the CHARMM36 and AMBER ff99SB-ILDNP force fields. The models that use implicit solvation methods were generally unreliable in predicting cysteine residue pKa's, with RMSDs between 3.41 and 4.72 pKa units. On average, the explicit solvent methods performed better than the implicit solvent methods. RMSD values of 2.40 and 3.20 were obtained for simulations with the CHARMM36 and AMBER ff99SB-ILDNP force fields, respectively. Further development of these methods is necessary because the performance of the best method is similar to that of the null-model (RMSD = 2.74) and these differences in RMSD are of limited statistical significance given the small size of our test set. PMID:27541839

  8. Roles of N-terminal pyroglutamate in maintaining structural integrity and pKa values of catalytic histidine residues in bullfrog ribonuclease 3.

    PubMed

    Lou, Yuan-Chao; Huang, Yu-Chie; Pan, Yun-Ru; Chen, Chinpan; Liao, You-Di

    2006-01-20

    Many proteins and bioactive peptides contain an N-terminal pyroglutamate residue (Pyr1). This residue reduces the susceptibility of the protein to aminopeptidases and often has important functional roles. The antitumor ribonuclease RC-RNase 3 (RNase 3) from oocytes of Rana catesbeiana (bullfrog) is one such protein. We have produced recombinant RNase 3 containing the N-terminal Pyr1 (pRNase 3) and found it to be indistinguishable from the native RNase 3 by mass spectrometry and a variety of other biochemical and immunological criteria. We demonstrated by NMR analysis that the Pyr1 of pRNase 3 forms hydrogen bonds with Lys9 and Ile96 and stabilizes the N-terminal alpha-helix in a rigid conformation. In contrast, the N-terminal alpha-helix becomes flexible and the pKa values of the catalytic residues His10 and His97 altered when Pyr1 formation is blocked by an extra methionine at the N terminus in the recombinant mqRNase 3. Thus, our results provide a mechanistic explanation on the essential role of Pyr1 in maintaining the structural integrity, especially at the N-terminal alpha-helix, and in providing the proper environment for the ionization of His10 and His97 residues for catalysis and cytotoxicity against HeLa cells.

  9. pK(a) prediction from "Quantum Chemical Topology" descriptors.

    PubMed

    Harding, A P; Wedge, D C; Popelier, P L A

    2009-08-01

    Knowing the pK(a) of a compound gives insight into many properties relevant to many industries, in particular the pharmaceutical industry during drug development processes. In light of this, we have used the theory of Quantum Chemical Topology (QCT), to provide ab initio descriptors that are able to accurately predict pK(a) values for 228 carboxylic acids. This Quantum Topological Molecular Similarity (QTMS) study involved the comparison of 5 increasingly more expensive levels of theory to conclude that HF/6-31G(d) and B3LYP/6-311+G(2d,p) provided an accurate representation of the compounds studies. We created global and subset models for the carboxylic acids using Partial Least Square (PLS), Support Vector Machines (SVM), and Radial Basis Function Neural Networks (RBFNN). The models were extensively validated using 4-, 7-, and 10-fold cross-validation, with the validation sets selected based on systematic and random sampling. HF/6-31G(d) in conjunction with SVM provided the best statistics when taking into account the large increase in CPU time required to optimize the geometries at the B3LYP/6-311+G(2d,p) level. The SVM models provided an average q(2) value of 0.886 and an RMSE value of 0.293 for all the carboxylic acids, a q(2) of 0.825 and RMSE of 0.378 for the ortho-substituted acids, a q(2) of 0.923 and RMSE of 0.112 for the para- and meta-substituted acids, and a q(2) of 0.906 and RMSE of 0.268 for the aliphatic acids. Our method compares favorably to ACD/Laboratories, VCCLAB, SPARC, and ChemAxon's pK(a) prediction software based of the RMSE calculated by the leave-one-out method.

  10. Boric acid, “carbonic” acid, and N-containing oxyacids in aqueous solution: Ab initio studies of structure, pKa, NMR shifts, and isotopic fractionations

    NASA Astrophysics Data System (ADS)

    Tossell, J. A.

    2005-12-01

    B(OH) 3 and CO 2 are acidic species of considerable geochemical importance, yet the microscopic nature of the acid dissociation reactions for these B and C species is not well understood. Quantum mechanical methods have recently been applied to the direct ab initio calculation of p Ka values for many organic and inorganic weak acids, but the B and C acids have not yet been considered in detail. In the present study, p Ka values are calculated quantum mechanically for the oxyacids B(OH) 3, H 2CO 3 and HNO 3, which have experimental first p Ka values of 9.2, 6.4 and -1.3, respectively. We calculate the gas-phase reaction free energies at the highly accurate CBS-QB3 ab initio quantum mechanical level and reaction free energies of hydration using a polarizable continuum method. Using a thermodynamic cycle corresponding to the simple dissociation process HA A - + H +, in aqueous solution, we calculate p Ka values of 21.6, 3.8 to 2.2 and -0.8 for the three oxyacids mentioned above, closely matching experiment only for HNO 3. The discrepancies with experiment arise from the more complex nature of the acid dissociation process for B(OH) 3, which involves the addition of H 2O to B(OH) 3 and formation of the B(OH) 4- anion, and from the instability of hypothetical H 2CO 3 compared to the proper hydrated reactant complex CO 2. . . H 2O. When the proper microscopic description of the reactants and products is used the calculated p Ka values for the three acids become 11.1, 7.2 and -0.8, in considerably better agreement with experiment for B(OH) 3 and CO 2. . . H 2O. Thus p Ka calculations using this approach are accurate enough to give information on the actual acid species present in solution and the details of their acid dissociation processes at the microscopic level. 11B and 13C-NMR chemical shifts are also calculated for the various species and compared to experiment. By comparison of our calculations with experiment it is apparent that the 13C-NMR chemical shift has

  11. Kinetics of an acid-base catalyzed reaction (aspartame degradation) as affected by polyol-induced changes in buffer pH and pK values.

    PubMed

    Chuy, S; Bell, L N

    2009-01-01

    The kinetics of an acid-base catalyzed reaction, aspartame degradation, were examined as affected by the changes in pH and pK(a) values caused by adding polyols (sucrose, glycerol) to phosphate buffer. Sucrose-containing phosphate buffer solutions had a lower pH than that of phosphate buffer alone, which contributed, in part, to reduced aspartame reactivity. A kinetic model was introduced for aspartame degradation that encompassed pH and buffer salt concentrations, both of which change with a shift in the apparent pK(a) value. Aspartame degradation rate constants in sucrose-containing solutions were successfully predicted using this model when corrections (that is, lower pH, lower apparent pK(a) value, buffer dilution from the polyol) were applied. The change in buffer properties (pH, pK(a)) from adding sucrose to phosphate buffer does impact food chemical stability. These effects can be successfully incorporated into predictive kinetic models. Therefore, pH and pK(a) changes from adding polyols to buffer should be considered during food product development.

  12. Novel Instrument for Automated pK(a) Determination by Internal Standard Capillary Electrophoresis.

    PubMed

    Cabot, Joan M; Fuguet, Elisabet; Rosés, Martí; Smejkal, Petr; Breadmore, Michael C

    2015-06-16

    The internal standard capillary electrophoresis method (IS-CE) has been implemented in a novel sequential injection-capillary electrophoresis instrument for the high-throughput determination of acidity constants (pK(a)) regardless of aqueous solubility, number of pK(a) values, or structure. This instrument comprises a buffer creation system that automatically mixes within a few seconds four reagents for in situ creation of the separation electrolyte with a pH range of 2-13, ionic strength of 10-100 mM and organic solvent content from 0% to 40%. Combined with 1.2 kV/cm and a short effective length (15 cm to the UV detector) fast 20 s electrophoretic separations can be obtained. The low standard deviation of the replicates and the low variation compared to reference values show that this system can accurately determine acidity constants of drugs by IS-CE. A single pK(a) can be determined in 2 min and a set of 20 measurements in half an hour, allowing rapid, simple, and flexible determination of pK(a) values of pharmaceutical targets.

  13. Internal standard capillary electrophoresis as a high-throughput method for pKa determination in drug discovery and development.

    PubMed

    Cabot, Joan M; Fuguet, Elisabet; Rosés, Martí

    2014-10-13

    A novel high-throughput method for determining acidity constants (pKa) by capillary electrophoresis (CE) is developed. The method, based on the use of an internal standard (IS-CE), is implemented as a routine method for accurate experimental pKa determination of drugs undergoing physicochemical measurements in drug discovery laboratories. Just two electropherograms at 2 different pH values are needed to calculate an acidity constant. Several ISs can be used in the same buffer and run to enhance precision. With 3 ISs, for example, the pKa of the test compound (TC) can be obtained in triplicate in less than 3 min of electrophoresis. It has been demonstrated that the IS-CE method eliminates some systematic errors, maintaining, or even increasing the precision of the results compared with other methods. Furthermore, pH buffer instability during electrophoretic runs is not a problem in the IS-CE method. It is also proved that after 16 h of electroseparation using the same buffer vial, pH may change by around one unit; but the pKa calculated by the IS-CE method remains constant. Thus, IS-CE is a powerful high-throughput method for pKa determination in drug discovery and development.

  14. Determination of pK(a) of felodipine using UV-Visible spectroscopy.

    PubMed

    Pandey, M M; Jaipal, A; Kumar, A; Malik, R; Charde, S Y

    2013-11-01

    In the present study, for the first time, experimental pKa value of felodipine is reported. Dissociation constant, pKa, is one of the very important physicochemical properties of drugs. It is of paramount significance from the perspective of pharmaceutical analysis and dosage form design. The method used for the pKa determination of felodipine was essentially a UV-Visible spectrophotometric method. The spectrophotometric method for the pKa determination was opted by acknowledging the established fact that spectrophotometric determination of pKa produces most precise values. The pKa of felodipine was found to be 5.07. Furthermore, the ruggedness of the determined value is also validated in this study in order to produce exact pKa of the felodipine. PMID:23906645

  15. Investigating the discrimination potential of linear and nonlinear spectral multivariate calibrations for analysis of phenolic compounds in their binary and ternary mixtures and calculation pKa values.

    PubMed

    Rasouli, Zolaikha; Ghavami, Raouf

    2016-08-01

    Vanillin (VA), vanillic acid (VAI) and syringaldehyde (SIA) are important food additives as flavor enhancers. The current study for the first time is devote to the application of partial least square (PLS-1), partial robust M-regression (PRM) and feed forward neural networks (FFNNs) as linear and nonlinear chemometric methods for the simultaneous detection of binary and ternary mixtures of VA, VAI and SIA using data extracted directly from UV-spectra with overlapped peaks of individual analytes. Under the optimum experimental conditions, for each compound a linear calibration was obtained in the concentration range of 0.61-20.99 [LOD=0.12], 0.67-23.19 [LOD=0.13] and 0.73-25.12 [LOD=0.15] μgmL(-1) for VA, VAI and SIA, respectively. Four calibration sets of standard samples were designed by combination of a full and fractional factorial designs with the use of the seven and three levels for each factor for binary and ternary mixtures, respectively. The results of this study reveal that both the methods of PLS-1 and PRM are similar in terms of predict ability each binary mixtures. The resolution of ternary mixture has been accomplished by FFNNs. Multivariate curve resolution-alternating least squares (MCR-ALS) was applied for the description of spectra from the acid-base titration systems each individual compound, i.e. the resolution of the complex overlapping spectra as well as to interpret the extracted spectral and concentration profiles of any pure chemical species identified. Evolving factor analysis (EFA) and singular value decomposition (SVD) were used to distinguish the number of chemical species. Subsequently, their corresponding dissociation constants were derived. Finally, FFNNs has been used to detection active compounds in real and spiked water samples. PMID:27176001

  16. Investigating the discrimination potential of linear and nonlinear spectral multivariate calibrations for analysis of phenolic compounds in their binary and ternary mixtures and calculation pKa values

    NASA Astrophysics Data System (ADS)

    Rasouli, Zolaikha; Ghavami, Raouf

    2016-08-01

    Vanillin (VA), vanillic acid (VAI) and syringaldehyde (SIA) are important food additives as flavor enhancers. The current study for the first time is devote to the application of partial least square (PLS-1), partial robust M-regression (PRM) and feed forward neural networks (FFNNs) as linear and nonlinear chemometric methods for the simultaneous detection of binary and ternary mixtures of VA, VAI and SIA using data extracted directly from UV-spectra with overlapped peaks of individual analytes. Under the optimum experimental conditions, for each compound a linear calibration was obtained in the concentration range of 0.61-20.99 [LOD = 0.12], 0.67-23.19 [LOD = 0.13] and 0.73-25.12 [LOD = 0.15] μg mL- 1 for VA, VAI and SIA, respectively. Four calibration sets of standard samples were designed by combination of a full and fractional factorial designs with the use of the seven and three levels for each factor for binary and ternary mixtures, respectively. The results of this study reveal that both the methods of PLS-1 and PRM are similar in terms of predict ability each binary mixtures. The resolution of ternary mixture has been accomplished by FFNNs. Multivariate curve resolution-alternating least squares (MCR-ALS) was applied for the description of spectra from the acid-base titration systems each individual compound, i.e. the resolution of the complex overlapping spectra as well as to interpret the extracted spectral and concentration profiles of any pure chemical species identified. Evolving factor analysis (EFA) and singular value decomposition (SVD) were used to distinguish the number of chemical species. Subsequently, their corresponding dissociation constants were derived. Finally, FFNNs has been used to detection active compounds in real and spiked water samples.

  17. On the development of protein pKa calculation algorithms

    SciTech Connect

    Carstensen, Tommy; Farrell, Damien; Huang, Yong; Baker, Nathan A.; Nielsen, Jens E.

    2011-12-01

    Protein pKa calculation algorithms are typically developed to reproduce experimental pKa values and provide us with a better understanding of the fundamental importance of electrostatics for protein structure and function. However, the approximations and adjustable parameters employed in almost all pKa calculation methods means that there is the risk that pKa calculation algorithms are 'over-fitted' to the available datasets, and that these methods therefore do not model protein physics realistically. We employ simulations of the protein pKa calculation algorithm development process to show that careful optimization procedures and non-biased experimental datasets must be applied to ensure a realistic description of the underlying physical terms. We furthermore investigate the effect of experimental noise and find a significant effect on the pKa calculation algorithm optimization landscape. Finally, we comment on strategies for ensuring the physical realism of protein pKa calculation algorithms and we assess the overall state of the field with a view to predicting future directions of development.

  18. Feedback regulation between autophagy and PKA

    PubMed Central

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA. PMID:26046386

  19. Feedback regulation between autophagy and PKA.

    PubMed

    Torres-Quiroz, Francisco; Filteau, Marie; Landry, Christian R

    2015-01-01

    Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

  20. Measurement and modeling of acid dissociation constants of tri-peptides containing Glu, Gly, and His using potentiometry and generalized multiplicative analysis of variance.

    PubMed

    Khoury, Rima Raffoul; Sutton, Gordon J; Hibbert, D Brynn; Ebrahimi, Diako

    2013-02-28

    We report pK(a) values with measurement uncertainties for all labile protons of the 27 tri-peptides prepared from the amino acids glutamic acid (E), glycine (G) and histidine (H). Each tri-peptide (GGG, GGE, GGH, …, HHH) was subjected to alkali titration and pK(a) values were calculated from triplicate potentiometric titrations data using HyperQuad 2008 software. A generalized multiplicative analysis of variance (GEMANOVA) of pK(a) values for the most acidic proton gave the optimum model having two terms, an interaction between the end amino acids plus an isolated main effect of the central amino acid.

  1. Measurement and modeling of acid dissociation constants of tri-peptides containing Glu, Gly, and His using potentiometry and generalized multiplicative analysis of variance.

    PubMed

    Khoury, Rima Raffoul; Sutton, Gordon J; Hibbert, D Brynn; Ebrahimi, Diako

    2013-02-28

    We report pK(a) values with measurement uncertainties for all labile protons of the 27 tri-peptides prepared from the amino acids glutamic acid (E), glycine (G) and histidine (H). Each tri-peptide (GGG, GGE, GGH, …, HHH) was subjected to alkali titration and pK(a) values were calculated from triplicate potentiometric titrations data using HyperQuad 2008 software. A generalized multiplicative analysis of variance (GEMANOVA) of pK(a) values for the most acidic proton gave the optimum model having two terms, an interaction between the end amino acids plus an isolated main effect of the central amino acid. PMID:23247603

  2. Acidity of Strong Acids in Water and Dimethyl Sulfoxide.

    PubMed

    Trummal, Aleksander; Lipping, Lauri; Kaljurand, Ivari; Koppel, Ilmar A; Leito, Ivo

    2016-05-26

    Careful analysis and comparison of the available acidity data of HCl, HBr, HI, HClO4, and CF3SO3H in water, dimethyl sulfoxide (DMSO), and gas-phase has been carried out. The data include experimental and computational pKa and gas-phase acidity data from the literature, as well as high-level computations using different approaches (including the W1 theory) carried out in this work. As a result of the analysis, for every acid in every medium, a recommended acidity value is presented. In some cases, the currently accepted pKa values were revised by more than 10 orders of magnitude. PMID:27115918

  3. Computational study on the acidic constants of chiral Brønsted acids in dimethyl sulfoxide.

    PubMed

    Yang, Chen; Xue, Xiao-Song; Li, Xin; Cheng, Jin-Pei

    2014-05-16

    The pK(a) values of a series of chiral Brønsted acids, including N-triflylphosphoramides, bis(sulfonyl)imides, bis(sulfuryl)imides, dicarboxylic acids, sulfonic acids, and N-phosphinyl phosphoramides, were predicted by using the SMD/M06-2x/6-311++G(2df,2p)//B3LYP/6-31+G(d) method in DMSO. The results revealed that the calculated pKa values ranged from -9.06 to 12.18 for different types of acids. The influence of acidic strength on reactivity and stereoselectivity was discussed using the calculated acidity data. Given that the choice of catalyst with appropriate acidity is the primary condition, several new catalyst candidates were designed by calculating corresponding pK(a) values of parent acids.

  4. Direct Raman measurement of an elevated base pKa in the active site of a small ribozyme in a precatalytic conformation.

    PubMed

    Guo, Man; Spitale, Robert C; Volpini, Rosaria; Krucinska, Jolanta; Cristalli, Gloria; Carey, Paul R; Wedekind, Joseph E

    2009-09-16

    Catalytic RNA molecules can achieve rate acceleration by shifting base pK(a) values toward neutrality. Prior evidence has suggested that base A38 of the hairpin ribozyme plays an important role in phosphoryl transfer, possibly functioning as a general acid, or by orienting a specific water molecule for proton transfer. To address the role of A38, we used Raman spectroscopy to measure directly the pK(a) of the N1-imino moiety in the context of hairpin ribozyme crystals representative of a "precatalytic" conformation. The results revealed that the pK(a) of A38 is shifted to 5.46 +/- 0.05 relative to 3.68 +/- 0.06 derived from a reference solution of the nucleotide AMP. The elevated pK(a) correlates well with the first titration point of the macroscopic pH-rate profile of the hairpin ribozyme in solution and strongly supports A38 as a general acid catalyst in bond scission. The results confirm that A38 is protonated before the transition state, which would promote phosphorane development. Overall, the results establish a cogent structure-function paradigm that expands our understanding of how RNA structure can enhance nucleobase reactivity to catalyze biological reactions.

  5. Aspartate embedding depth affects pHLIP's insertion pKa.

    PubMed

    Fendos, Justin; Barrera, Francisco N; Engelman, Donald M

    2013-07-01

    We have used the pHlow insertion peptide (pHLIP) family to study the role of aspartate embedding depth in pH-dependent transmembrane peptide insertion. pHLIP binds to the surface of a lipid bilayer as a largely unstructured monomer at neutral pH. When the pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when the pH is adjusted back to a neutral value. One of the critical events facilitating pHLIP insertion is the protonation of aspartates in the spanning domain of the peptide: the negative side chains of these residues convert to uncharged, polar forms, facilitating insertion by altering the hydrophobicity of the spanning domain. To examine this protonation mechanism further, we created pHLIP sequence variants in which the two spanning aspartates (D14 and D25) were moved up or down in the sequence. We hypothesized that the aspartate depth in the inserted state would directly affect the proton affinity of the acidic side chains, altering the pKa of pH-dependent insertion. To this end, we also mutated the arginine at position 11 to determine whether arginine snorkeling modulates the insertion pKa by affecting the aspartate depth. Our results indicate that both types of mutations change the insertion pKa, supporting the idea that the aspartate depth is a participating parameter in determining the pH dependence. We also show that pHLIP's resistance to aggregation can be altered with our mutations, identifying a new criterion for improving the performance of pHLIP in vivo when targeting acidic disease tissues such as cancer and inflammation. PMID:23721379

  6. The vasorelaxant effect of 8(17),12E,14-labdatrien-18-oic acid involves stimulation of adenylyl cyclase and cAMP/PKA pathway: Evidences by pharmacological and molecular docking studies.

    PubMed

    Ribeiro, Luciano A A; Alencar Filho, Edilson B; Coelho, Maisa C; Silva, Bagnólia A

    2015-10-01

    The relaxant effect of 8(17),12E,14-labdatrien-18-oic acid (LBD) was investigated on isolated aortic rings and compared with forskolin (FSK), a standard and potent activator of adenylyl cyclase (AC) with relaxing effect. The presence of potassium channel blockers, such as glibenclamide (ATP-blocker), apamin (SKCa-blocker), charybdotoxin (BKCa-blocker) did not significantly affect either the LBD or FSK concentration-response curves. However, in the presence of 4-aminopyridine (KV-blocker), the relaxant effect for both diterpenes was significantly attenuated, with reduction of its relative potencies. Moreover, the relaxation induced by 8-Br-cAMP, an analog of cAMP, was also significantly attenuated in the same conditions, i.e., in the presence of 4-aminopyridine. The presence of aminophylline, a nonselective phosphodiesterase inhibitor, caused a significant increasing in the potency for both LBD and FSK. On the other hand, the presence of Rp-cAMPS, a selective PKA-inhibitor, significantly attenuated the relaxant effect of LBD. In this work, in the same experimental conditions, both labdane-type diterpenes presented remarkably similar results; FSK, however, presented a higher potency (100-fold) than LBD. Thus, the hypothesis that LBD could be a novel AC-activator emerged. To assess that hypothesis, computational molecular docking studies were performed. Crystallographic structure of adenylyl cyclase/forskolin complex (1AB8) was obtained from RSCB Protein Data Bank and used to compare the modes of interaction of the native ligand and LBD. The computational data shows many similarities between LBD and FSK concerning the interaction with the regulatory site of AC. Taken together, the results presented here pointed to LBD as a novel AC-activator. PMID:26144373

  7. Nitrosyl-centered redox and acid-base interconversions in [Ru(Me3[9]aneN3)(bpy)(NO)](3,2,1+). The pKa of HNO for its nitroxyl derivative in aqueous solution.

    PubMed

    Codesido, Nicolás Osa; Weyhermüller, Thomas; Olabe, José A; Slep, Leonardo D

    2014-01-21

    This work reports the preparation of a new 6-coordinated nitrosyl compound and its use as a model to explore the redox and acid-base properties of the three redox states of bound nitrosyl (formally NO(+), NO(•), NO(-)/HNO) in {RuNO}(6,7,8) species. We prepared the octahedral {RuNO}(6) complex [Ru(Me3[9]aneN3)(bpy)(NO)](3+) (Me3[9]aneN3: 1,4,7-trimethyl-1,4,7-triazacyclononane; bpy = 2,2'-bipyridine), and the related [Ru(Me3[9]aneN3)(bpy)(NO2)](+) nitro derivative. The compounds were characterized by chemical analysis, X-ray diffraction, NMR, IR, and UV-vis spectroscopies, cyclic voltammetry (CV), UV-vis/IR spectroelectrochemistry, and theoretical calculations (DFT, (TD)DFT). The reaction kinetics between the {RuNO}(6) complex and the nucleophile OH(-) is also presented. The incorporation of tridentate and bidentate ligands in the coordination sphere prevents labilization issues associated with the trans effect when attaining the reduced states of the nitrosyl group. This allows for a consistent interpretation of the changes in the main geometrical parameters: Ru-N and N-O distances, Ru-N-O angle, and the νNO frequency and electronic transitions. We explore the redox properties in acetonitrile and aqueous solutions, and provide a potential (E1/2) - pH (Pourbaix) diagram for the three diatomic nitrosyl-bound species, as well as for HNO and NO2(-), including the report of the pKa of the [Ru(Me3[9]aneN3)(bpy)(HNO)](2+) ion, 9.78 ± 0.15 at 25.0 °C.

  8. Fast high-throughput method for the determination of acidity constants by capillary electrophoresis. II. Acidic internal standards.

    PubMed

    Cabot, Joan Marc; Fuguet, Elisabet; Ràfols, Clara; Rosés, Martí

    2010-12-24

    A fast method for the determination of acidity constants by CZE has been recently developed. This method is based on the use of an internal standard of pK(a) similar to that of the analyte. In this paper we establish the reference pK(a) values of a set of 24 monoprotic neutral acids of varied structure that we propose as internal standards. These compounds cover the most usual working pH range in CZE and facilitate the selection of adequate internal standards for a given determination. The reference pK(a) values of the acids have been established by the own internal standard method, i.e. from the mobility differences between different acids of similar pK(a) in the same pH buffers. The determined pK(a) values have been contrasted to the literature pK(a) values and confirmed by determination of the pK(a) values of some acids of the set by the classical CE method. Some systematic deviations of mobilities have been observed in NaOH buffer in reference to the other used buffers, overcoming the use of NaOH in the classical CE method. However, the deviations affect in a similar degree to the test compounds and internal standards allowing thus, the use of NaOH buffer in the internal standard method. This fact demonstrates the better performance of the internal standard method over the classical method to correct mobility deviations, which together with its fastness makes it an interesting method for the routine determination of accurate pK(a) values of new pharmaceutical drugs and drug precursors.

  9. pKa at Quartz/Electrolyte Interfaces.

    PubMed

    Pfeiffer-Laplaud, Morgane; Gaigeot, Marie-Pierre; Sulpizi, Marialore

    2016-08-18

    Acidity of silanol sites at the crystalline quartz/aqueous electrolyte (NaCl, NaI, KCl) interfaces are calculated from ab initio molecular dynamics simulations. pKa's are found to follow a combination of the cationic and anionic Hofmeister series in the order pKa(neat solution) < pKa(NaCl) < pKa(NaI) < pKa(KCl), in agreement with experimental measurements. Rationalization of this ranking is achieved in terms of the microscopic local solvation of the protonated silanols and their conjugated bases, the silanolates SiO(-). The change in the pKa is the result of both water destructuring by alkali halides, as well as of the specific cation/SiO(-) interaction, depending on the electrolyte. Molecular modeling at the atomistic level is required to achieve such comprehension, with ab initio molecular dynamics being able to model complex inhomogeneous charged interfaces and the associated interfacial chemical reactivity. PMID:27483195

  10. First-principles calculation of pKa for cocaine, nicotine, neurotransmitters, and anilines in aqueous solution.

    PubMed

    Lu, Haiting; Chen, Xi; Zhan, Chang-Guo

    2007-09-01

    The absolute pKa values of 24 representative amine compounds, including cocaine, nicotine, 10 neurotransmitters, and 12 anilines, in aqueous solution were calculated by performing first-principles electronic structure calculations that account for the solvent effects using four different solvation models, i.e., the surface and volume polarization for electrostatic interaction (SVPE) model, the standard polarizable continuum model (PCM), the integral equation formalism for the polarizable continuum model (IEFPCM), and the conductor-like screening solvation model (COSMO). Within the examined computational methods, the calculations using the SVPE model lead to the absolute pKa values with the smallest root-mean-square-deviation (rmsd) value (1.18). When the SVPE model was replaced by the PCM, IEFPCM, and COSMO, the rmsd value of the calculated absolute pKa values became 3.21, 2.72, and 3.08, respectively. All types of calculated pKa values linearly correlate with the experimental pKa values very well. With the empirical corrections using the linear correlation relationships, the theoretical pKa values are much closer to the corresponding experimental data and the rmsd values become 0.51-0.83. The smallest rmsd value (0.51) is also associated with the SVPE model. All of the results suggest that the first-principles electronic structure calculations using the SVPE model are a reliable approach to the pKa prediction for the amine compounds. PMID:17691837

  11. First-principles calculation of pKa for cocaine, nicotine, neurotransmitters, and anilines in aqueous solution

    PubMed Central

    Lu, Haiting; Chen, Xi; Zhan, Chang-Guo

    2010-01-01

    The absolute pKa values of 24 representative amine compounds, including cocaine, nicotine, 10 neurotransmitters, and 12 anilines, in aqueous solution were calculated by performing first-principles electronic structure calculations that account for the solvent effects using four different solvation models, i.e. the surface and volume polarization for electrostatic interaction (SVPE) model, the standard polarizable continuum model (PCM), the integral equation formalism for the polarizable continuum model (IEFPCM), and the conductor-like screening solvation model (COSMO). Within the examined computational methods, the calculations using the SVPE model lead to the absolute pKa values with the smallest root-mean-square-deviation (RMSD) value (1.18). When the SVPE model was replaced by the PCM, IEFPCM, and COSMO, the RMSD value of the calculated absolute pKa values became 3.21, 2.72, and 3.08, respectively. All types of calculated pKa values linearly correlate with the experimental pKa values very well. With the empirical corrections using the linear correlation relationships, the theoretical pKa values are much closer to the corresponding experimental data and the RMSD values become 0.51 to 0.83. The smallest RMSD value (0.51) is also associated with the SVPE model. All of the results suggest that the first-principles electronic structure calculations using the SVPE model are a reliable approach to the pKa prediction for the amine compounds. PMID:17691837

  12. PKA regulatory subunit expression in tooth development.

    PubMed

    de Sousa, Sílvia Ferreira; Kawasaki, Katsushige; Kawasaki, Maiko; Volponi, Ana Angelova; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri; Sharpe, Paul T; Ohazama, Atsushi

    2014-05-01

    Protein kinase A (PKA) plays critical roles in many biological processes including cell proliferation, cell differentiation, cellular metabolism and gene regulation. Mutation in PKA regulatory subunit, PRKAR1A has previously been identified in odontogenic myxomas, but it is unclear whether PKA is involved in tooth development. The aim of the present study was to assess the expression of alpha isoforms of PKA regulatory subunit (Prkar1a and Prkar2a) in mouse and human odontogenesis by in situ hybridization. PRKAR1A and PRKAR2A mRNA transcription was further confirmed in a human deciduous germ by qRT-PCR. Mouse Prkar1a and human PRKAR2A exhibited a dynamic spatio-temporal expression in tooth development, whereas neither human PRKAR1A nor mouse Prkar2a showed their expression in odontogenesis. These isoforms thus showed different expression pattern between human and mouse tooth germs. PMID:24755349

  13. PKA regulatory subunit expression in tooth development.

    PubMed

    de Sousa, Sílvia Ferreira; Kawasaki, Katsushige; Kawasaki, Maiko; Volponi, Ana Angelova; Gomez, Ricardo Santiago; Gomes, Carolina Cavaliéri; Sharpe, Paul T; Ohazama, Atsushi

    2014-05-01

    Protein kinase A (PKA) plays critical roles in many biological processes including cell proliferation, cell differentiation, cellular metabolism and gene regulation. Mutation in PKA regulatory subunit, PRKAR1A has previously been identified in odontogenic myxomas, but it is unclear whether PKA is involved in tooth development. The aim of the present study was to assess the expression of alpha isoforms of PKA regulatory subunit (Prkar1a and Prkar2a) in mouse and human odontogenesis by in situ hybridization. PRKAR1A and PRKAR2A mRNA transcription was further confirmed in a human deciduous germ by qRT-PCR. Mouse Prkar1a and human PRKAR2A exhibited a dynamic spatio-temporal expression in tooth development, whereas neither human PRKAR1A nor mouse Prkar2a showed their expression in odontogenesis. These isoforms thus showed different expression pattern between human and mouse tooth germs.

  14. An Isoform specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes

    PubMed Central

    Zhang, Ping; Ye, Feng; Bastidas, Adam C.; Kornev, Alexandr P.; Wu, Jian; Ginsberg, Mark H.; Taylor, Susan S.

    2015-01-01

    SUMMARY cAMP-dependent protein kinase (PKA) is regulated in part by N-terminal myristylation of its catalytic (C) subunit. Structural information about the role of myristylation in membrane targeting of PKA has been limited. In mammalian cells there are four functionally non-redundant PKA Regulatory subunits (RIα, RIβ, RIIα, and RIIβ). PKA is assembled as an inactive R2C2 holoenzyme in cells. To explore the role of N-myristylation in membrane targeting of PKA holoenzymes, we solved crystal structures of RIα:myrC and RIIβ2:myrC2 and showed that the N-terminal myristylation site in the myrC serves as a flexible “switch” that can potentially be mobilized for membrane anchoring of RII, but not RI, holoenzymes. Furthermore, we synthesized nanodiscs and showed by electron microscopy that membrane targeting through the myristic acid is specific for the RII holoenzyme. This membrane anchoring myristylation switch is independent of A Kinase Anchoring Proteins (AKAPs) that target PKA to membranes by other mechanisms. PMID:26278174

  15. Determination of acid dissociation constants of warfarin and hydroxywarfarins by capillary electrophoresis.

    PubMed

    Nowak, Paweł; Olechowska, Paulina; Mitoraj, Mariusz; Woźniakiewicz, Michał; Kościelniak, Paweł

    2015-08-10

    In this work the acid dissociation constants--pKa of warfarin and its all important oxidative metabolites have been determined by capillary electrophoresis-based methods. It has resulted in a complete description of two acid-base dissociation equilibria, yet not investigated experimentally for phase I metabolites of warfarin. The capillary electrophoresis (CE) method based on the relation between effective electrophoretic mobilities and pH has proven to be a suitable tool for pKa determination, while the spectrophotometric (CE-DAD) and the internal standard methods (IS-CE), have appeared to be promising alternative approaches. The CE-DAD approach based on the change in absorbance spectra between the acidic and basic forms is a combination between capillary electrophoresis and spectrophotometric titration, and yields very consistent values of pKa1 with CE. The IS-CE, in turn, enables an estimation of pKa1 and pKa2 from only two analytical runs, however, less accurate than CE and CE-DAD. The Debye-Hückel model has been confirmed experimentally as a good predictor of pKa values at various ionic strengths. Therefore, it has been used in determination of thermodynamic pKa1 and pKa2, referring to the zero ionic strength. The results are important from the analytical, pharmacological, and theoretical points of view.

  16. Determination of acid dissociation constants of warfarin and hydroxywarfarins by capillary electrophoresis.

    PubMed

    Nowak, Paweł; Olechowska, Paulina; Mitoraj, Mariusz; Woźniakiewicz, Michał; Kościelniak, Paweł

    2015-08-10

    In this work the acid dissociation constants--pKa of warfarin and its all important oxidative metabolites have been determined by capillary electrophoresis-based methods. It has resulted in a complete description of two acid-base dissociation equilibria, yet not investigated experimentally for phase I metabolites of warfarin. The capillary electrophoresis (CE) method based on the relation between effective electrophoretic mobilities and pH has proven to be a suitable tool for pKa determination, while the spectrophotometric (CE-DAD) and the internal standard methods (IS-CE), have appeared to be promising alternative approaches. The CE-DAD approach based on the change in absorbance spectra between the acidic and basic forms is a combination between capillary electrophoresis and spectrophotometric titration, and yields very consistent values of pKa1 with CE. The IS-CE, in turn, enables an estimation of pKa1 and pKa2 from only two analytical runs, however, less accurate than CE and CE-DAD. The Debye-Hückel model has been confirmed experimentally as a good predictor of pKa values at various ionic strengths. Therefore, it has been used in determination of thermodynamic pKa1 and pKa2, referring to the zero ionic strength. The results are important from the analytical, pharmacological, and theoretical points of view. PMID:25968611

  17. Discovery of Allostery in PKA Signaling

    PubMed Central

    Zhang, Ping; Kornev, Alexandr P.; Wu, Jian; Taylor, Susan S.

    2015-01-01

    cAMP-dependent protein kinase (PKA) was the second protein kinase to be discovered and the PKA catalytic (C) subunit serves as a prototype for the large protein kinase superfamily that contains over 500 gene products. The protein kinases regulate much of biology in eukaryotic cells and they are now also a major therapeutic target. Although PKA was discovered nearly 50 years ago and the subsequent discovery of the regulatory subunits that bind cAMP and release the catalytic activity from the holoenzyme followed quickly. Thus in PKA we see the convergence of two major signaling mechanisms - protein phosphorylation and second messenger signaling through cAMP. Crystallography provides a foundation for understanding function, and the structure of the isolated regulatory (R) and C-subunits have been extremely informative. Yet it is the R2C2 holoenzyme that predominates in cells, and one can only appreciate the allosteric features of PKA signaling by seeing the full length protein. The symmetry and the quaternary constraints that one R:C hetero-dimer exerts on the other in the holoenzyme simply are not present in the isolated subunits or even in the R:C hetero-dimer. PMID:26097522

  18. Potentiometric determination of pK(A) of organic bases in acetone by the application of coulometry.

    PubMed

    Vajgand, V J; Mihajlović, R P; Dzudović, R M

    1989-11-01

    A coulometric-potentiometric method for the determination of pK(A) values of organic bases in anhydrous acetone is described. The bases were titrated with protons obtained by anodic oxidation of hydrogen dissolved in palladium, in the presence of sodium perchlorate as the supporting electrolyte. A pair of glass electrodes was used for measuring directly the difference between the half-neutralization potentials of the standard and the base being studied. The pK(A) values obtained were close to those reported in the literature. The effect of the supporting electrolyte concentration on the pK(A) values of some of the bases was also studied.

  19. Hypothalamic PKA regulates leptin sensitivity and adiposity

    PubMed Central

    Yang, Linghai; McKnight, G. Stanley

    2015-01-01

    Mice lacking the RIIβ regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) display reduced adiposity and resistance to diet-induced obesity. Here we show that RIIβ knockout (KO) mice have enhanced sensitivity to leptin's effects on both feeding and energy metabolism. After administration of a low dose of leptin, the duration of hypothalamic JAK/STAT3 signalling is increased, resulting in enhanced POMC mRNA induction. Consistent with the extended JAK/STAT3 activation, we find that the negative feedback regulator of leptin receptor signalling, Socs3, is inhibited in the hypothalamus of RIIβ KO mice. During fasting, RIIβ–PKA is activated and this correlates with an increase in CREB phosphorylation. The increase in CREB phosphorylation is absent in the fasted RIIβ KO hypothalamus. Selective inhibition of PKA activity in AgRP neurons partially recapitulates the leanness and resistance to diet-induced obesity of RIIβ KO mice. Our findings suggest that RIIβ–PKA modulates the duration of leptin receptor signalling and therefore the magnitude of the catabolic response to leptin. PMID:26381935

  20. PKA signaling drives mammary tumorigenesis through Src.

    PubMed

    Beristain, A G; Molyneux, S D; Joshi, P A; Pomroy, N C; Di Grappa, M A; Chang, M C; Kirschner, L S; Privé, G G; Pujana, M A; Khokha, R

    2015-02-26

    Protein kinase A (PKA) hyperactivation causes hereditary endocrine neoplasias; however, its role in sporadic epithelial cancers is unknown. Here, we show that heightened PKA activity in the mammary epithelium generates tumors. Mammary-restricted biallelic ablation of Prkar1a, which encodes for the critical type-I PKA regulatory subunit, induced spontaneous breast tumors characterized by enhanced type-II PKA activity. Downstream of this, Src phosphorylation occurs at residues serine-17 and tyrosine-416 and mammary cell transformation is driven through a mechanism involving Src signaling. The phenotypic consequences of these alterations consisted of increased cell proliferation and, accordingly, expansion of both luminal and basal epithelial cell populations. In human breast cancer, low PRKAR1A/high SRC expression defines basal-like and HER2 breast tumors associated with poor clinical outcome. Together, the results of this study define a novel molecular mechanism altered in breast carcinogenesis and highlight the potential strategy of inhibiting SRC signaling in treating this cancer subtype in humans. PMID:24662820

  1. Conformational and oligomeric effects on the cysteine pK(a) of tryparedoxin peroxidase.

    PubMed

    Yuan, Ye; Knaggs, Michael H; Poole, Leslie B; Fetrow, Jacquelyn S; Salsbury, Freddie R

    2010-08-01

    Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (C(p)) supported by a lowered pK(a) in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decameric forms of one typical 2-Cys Prx, the tryparedoxin peroxidase from Trypanosoma cruzi (PDB id, 1uul). The calculations indicate that C(p) (C52) pK(a) values are highly affected by oligomeric state; an unshifted C(p) pK(a) (approximately 8.3, comparable to the pK(a) of isolated cysteine) is calculated for the monomer. In the dimers, starting with essentially identical structures, the C(p)s evolve dynamically asymmetric pK(a)s during the simulations; one subunit's C(p) pK(a) is shifted downward at a time, while the other is unshifted. However, when averaged over time, or multiple simulations, the two subunits within a dimer exhibit the same C(p), showing no preference for a lowered pK(a) in either subunit. Two conserved pathways that communicate the asymmetric pK(a)s between C(p)s of different subunits can be identified. In the decamer, all the C(p) pK(a)s are shifted downward, with slight asymmetry in the dimers which form the decamers. Structural analyses implicate oligomerization effects as responsible for these oligomeric state-dependent C(p) pK(a) shifts. The intra-dimer and the inter-dimer subunit contacts in the decamer restrict the conformations of the side chains of several residues (T49, T54 and E55) calculated to be key in shifting the C(p) pK(a). In addition, the backbone fluctuations of a few residues (M46, D47 and F48) result in a different electrostatic environment for the C(p) in dimers relative to the monomers. These side chain and backbone interactions

  2. Conformational and oligomeric effects on the cysteine pKa of typaredoxin peroxidase

    PubMed Central

    Yuan, Ye; Knaggs, Michael H.; Poole, Leslie B.; Fetrow, Jacquelyn S.; Salsbury, Freddie R.

    2010-01-01

    Typical 2-Cys peroxiredoxins (Prxs) are peroxidases which regulate cell signaling pathways, apoptosis, and differentiation. These enzymes are obligate homodimers, and can form decamers in solution. During catalysis, Prxs exhibit cysteine-dependent reactivity which requires the deprotonation of the peroxidatic cysteine (Cp) supported by a lowered pKa in the initial step. We present the results of molecular dynamics simulations combined with pKa calculations on the monomeric, dimeric and decameric forms of one typical 2-Cys Prx, the tryparedoxin peroxidase from Trypanosoma cruzi (PDB id, 1uul). The calculations indicate that Cp (C52) pKa values are highly affected by oligomeric state; an unshifted Cp pKa (~ 8.3, comparable to the pKa of isolated cysteine) is calculated for the monomer. In the dimers, starting with essentially identical structures, the Cps evolve dynamically asymmetric pKas during the simulations; one subunit’s Cp pKa is shifted downward at a time, while the other is unshifted. However, when averaged over time, or multiple simulations, the two subunits within a dimer exhibit the same Cp, showing no preference for a lowered pKa in either subunit. Two conserved pathways that communicate the asymmetric pKas between Cps of different subunits can be identified. In the decamer, all the Cp pKas are shifted downward, with slight asymmetry in the dimers which form the decamers. Structural analyses implicate oligomerization effects as responsible for these oligomeric state-dependent Cp pKa shifts. The intra-dimer and the inter-dimer subunit contacts in the decamer restrict the conformations of the side chains of several residues (T49, T54 and E55) calculated to be key in shifting the Cp pKa. In addition, the backbone fluctuations of a few residues (M46, D47 and F48) result in a different electrostatic environment for the Cp in dimers relative to the monomers. These side chain and backbone interactions which contribute to pKa modulation indicate the importance

  3. Acidosis Mediates the Switching of Gs-PKA and Gi-PKCε Dependence in Prolonged Hyperalgesia Induced by Inflammation

    PubMed Central

    Huang, Wei-Yu; Dai, Shih-Ping; Chang, Yan-Ching; Sun, Wei-Hsin

    2015-01-01

    Chronic inflammatory pain, when not effectively treated, is a costly health problem and has a harmful effect on all aspects of health-related quality of life. Previous studies suggested that in male Sprague Dawley rats, prostaglandin E2 (PGE2)-induced short-term hyperalgesia depends on protein kinase A (PKA) activity, whereas long-lasting hyperalgesia induced by PGE2 with carrageenan pre-injection, requires protein kinase Cε (PKCε). However, the mechanism underlying the kinase switch with short- to long-term hyperalgesia remains unclear. In this study, we used the inflammatory agents carrageenan or complete Freund’s adjuvant (CFA) to induce long-term hyperalgesia, and examined PKA and PKCε dependence and switching time. Hyperalgesia induced by both agents depended on PKA/PKCε and Gs/Gi-proteins, and the switching time from PKA to PKCε and from Gs to Gi was about 3 to 4 h after inflammation induction. Among the single inflammatory mediators tested, PGE2 and 5-HT induced transient hyperalgesia, which depended on PKA and PKCε, respectively. Only acidic solution-induced hyperalgesia required Gs-PKA and Gi-PKCε, and the switch time for kinase dependency matched inflammatory hyperalgesia, in approximately 2 to 4 h. Thus, acidosis in inflamed tissues may be a decisive factor to regulate switching of PKA and PKCε dependence via proton-sensing G-protein–coupled receptors. PMID:25933021

  4. Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans.

    PubMed

    Chowdhury, Tahmeena; Köhler, Julia R

    2015-10-01

    TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P-S6) was defined as a surrogate marker for TOR-dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P-S6 levels. The P-S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P-S6 to physiologic levels, whereas all four tested components were required to downregulate P-S6 in low glucose. TOR was epistatic to PKA components with respect to P-S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR-dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant.

  5. Determination of acid dissociation constants of triazole fungicides by pressure assisted capillary electrophoresis.

    PubMed

    Konášová, Renáta; Dytrtová, Jana Jaklová; Kašička, Václav

    2015-08-21

    Pressure assisted capillary electrophoresis was applied to determination of acid dissociation constants (pKa) of six widely used triazole fungicides (cyproconazole, epoxiconazole, flusilazole, tebuconazole, penconazole and propiconazole) in aqueous medium. The pKa values were determined from the dependence of effective electrophoretic mobility of the triazole fungicides on p[H(+)] of the background electrolyte (BGE) using non-linear regression analysis. The p[H(+)] was used instead of pH to reflect the increased ionic strength of the strongly acidic BGEs (pH<1.75) as compared to the BGEs at pH equal to or greater than 1.75. Prior to the pKa calculation, the measured effective electrophoretic mobilities were corrected to the reference temperature (25°C) and constant ionic strength (25mM). The regression function was modified to allow the determination of pKa in the BGEs of varying ionic strength. The electrophoretic measurements showed that the above triazole fungicides are very weak bases - their pKa values were found to be in the range 1.05-1.97 and were in a good agreement with the values calculated by SPARC online pKa calculator.

  6. [Degradation kinetics of chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid at neutral and alkaline pH values].

    PubMed

    Zhu, Peng; Miao, Xiao-lei; Chen, Yong

    2016-01-01

    The degradation kinetics of chlorogenic acid (5-CQA), cryptochlorogenic acid (4-CQA), and neochlorogenic acid (3-CQA) in aqueous solution at 37 degrees C and different pH values (7.05, 7.96, 9.25) were investigated in the present work. The results indicated that 3-, 4- and 5-CQA tended to remain stable in acidic pH circumstance, and unstable in neutral and alkaline pH circumstance. With the increase of the alkalinity, the degradation of 3-, 4- and 5-CQA was increased leading to a less amount of total CQA and was satisfactorily described by the Weibull equation. Meanwhile, caffeic acid was not detected after the degradation of CQA. Moreover, the degradation of 3-CQA and 5-CQA tended to be converted to 4-CQA, and the degradation of 4-CQA tended to be converted to 3-CQA rather than 5-CQA. The comparison of the degradation kinetics parameters of 3-, 4- and 5-CQA at neutral and alkaline pH values showed that the orders of the rate constant (k) values were 4-CQA > 3-CQA > 5-CQA, while the orders of the degradation half life (t½) values were 4-CQA < 3-CQA < 5-CQA, indicating the orders of the stabilities of 3-, 4- and 5-CQA at 37 degrees C and neutral and alkaline pH values were 4-CQA < 3-CQA < 5-CQA. PMID:27405173

  7. Discrimination of organic acids using a three molecule array based upon cruciform fluorophores.

    PubMed

    Davey, Evan A; Zucchero, Anthony J; Trapp, Oliver; Bunz, Uwe H F

    2011-05-25

    A small array was obtained from three reactive cruciform fluorophores in six different solvents. The array discerned 10 different aromatic carboxylic acids by protonation-induced fluorescence shifts, which were recorded by digital photography. This simple array can discern acids that have closely spaced pK(a) values.

  8. Enthalpies and constants of dissociation of several neutral and cationic acids in aqueous and methanol/water solutions at various temperatures.

    PubMed

    Shoghi, Elham; Romero, Lilian; Reta, Mario; Ràfols, Clara; Bosch, Elisabeth

    2009-05-01

    The acidic dissociation enthalpies and constants of anilinium, protonated tris(hydroxymethyl)aminomethane (HTris(+)), benzoic and acetic acids, have been determined at several temperatures in pure water and in methanol/water mixtures by potentiometry and by isothermal titration microcalorimetry (ITC). The pK(a) values determined by both techniques are in accordance when the dissociation process involves large amounts of heat. However, for the neutral acids the ITC technique gave slightly lower pK(a) values than those from potentiometry at the highest temperatures studied due to the small amounts of heat involved in the acidic dissociation. The dissociation enthalpies have been determined directly by calorimetry and the obtained values slightly decrease with the increase of temperature. Therefore, only a rough estimation of the dissociation enthalpies can be obtained from potentiometric pK(a) by means of the Van't Hoff approach.

  9. Enthalpies and constants of dissociation of several neutral and cationic acids in aqueous and methanol/water solutions at various temperatures.

    PubMed

    Shoghi, Elham; Romero, Lilian; Reta, Mario; Ràfols, Clara; Bosch, Elisabeth

    2009-05-01

    The acidic dissociation enthalpies and constants of anilinium, protonated tris(hydroxymethyl)aminomethane (HTris(+)), benzoic and acetic acids, have been determined at several temperatures in pure water and in methanol/water mixtures by potentiometry and by isothermal titration microcalorimetry (ITC). The pK(a) values determined by both techniques are in accordance when the dissociation process involves large amounts of heat. However, for the neutral acids the ITC technique gave slightly lower pK(a) values than those from potentiometry at the highest temperatures studied due to the small amounts of heat involved in the acidic dissociation. The dissociation enthalpies have been determined directly by calorimetry and the obtained values slightly decrease with the increase of temperature. Therefore, only a rough estimation of the dissociation enthalpies can be obtained from potentiometric pK(a) by means of the Van't Hoff approach. PMID:19261425

  10. Strong-acid, carboxyl-group structures in fulvic acid from the Suwannee River, Georgia. 2. Major structures

    USGS Publications Warehouse

    Leenheer, J.A.; Wershaw, R. L.; Reddy, M.M.

    1995-01-01

    Polycarboxylic acid structures that account for the strong-acid characteristics (pKa1 near 2.0) were examined for fulvic acid from the Suwannee River. Studies of model compounds demonstrated that pKa values near 2.0 occur only if the ??-ether or ??-ester groups were in cyclic structures with two to three additional electronegative functional groups (carboxyl, ester, ketone, aromatic groups) at adjacent positions on the ring. Ester linkage removal by alkaline hydrolysis and destruction of ether linkages through cleavage and reduction with hydriodic acid confirmed that the strong carboxyl acidity in fulvic acid was associated with polycarboxylic ??-ether and ??-ester structures. Studies of hypothetical structural models of fulvic acid indicated possible relation of these polycarboxylic structures with the amphiphilic and metal-binding properties of fulvic acid.

  11. Determination of acid dissociation constant of 20 coumarin derivatives by capillary electrophoresis using the amine capillary and two different methodologies.

    PubMed

    Nowak, Paweł Mateusz; Woźniakiewicz, Michał; Piwowarska, Monika; Kościelniak, Paweł

    2016-05-13

    In this work capillary electrophoresis has been used to determine acid dissociation constant of 20 structurally diverse coumarin derivatives. For a majority of compounds pKa value has been determined for the first time. The obtained values vary between 4.16-9.10pH unit, pointing to the interesting structure-acidity relationships. The amine permanently coated capillary has been applied for that purpose, because it has turned out to be more effective in pKa determination than the bare silica and other coated capillaries, ensuring good precision and shorter migration times. A traditional methodology relying on measurements in a broad pH range and fitting of a sigmoidal function has been compared to an alternative simplified approach, reported for the first time, where only two electrophoretic mobility values suffice for pKa estimation. The first value corresponds to the partially ionized form and it is measured experimentally, while the second one to the totally ionized form - it is measured experimentally (two-values method) or estimated directly from molecular mass (one-value method). We show that despite a limited measurements number, the alternative approach may be consistent with the traditional methodology, yielding the relatively low pKa deviation. Its reliability has also been confirmed by the analytical predictions, comprising resolution, migration order, migration times and peaks overlapping. Therefore, combination of the amine capillary with the simplified calculation method is an attractive tool for fast and reliable pKa estimation.

  12. More value from food waste: Lactic acid and biogas recovery.

    PubMed

    Kim, Mi-Sun; Na, Jeong-Geol; Lee, Mo-Kwon; Ryu, Hoyoung; Chang, Yong-Keun; Triolo, Jin M; Yun, Yeo-Myeong; Kim, Dong-Hoon

    2016-06-01

    Anaerobic digestion (AD) is one of the traditional technologies for treating organic solid wastes, but its economic benefit is sometimes questioned. To increase the economic feasibility of the treatment process, the aim of this study was to recover not only biogas from food waste but lactic acid (LA) as well. At first, LA fermentation of food waste (FW) was conducted using an indigenous mixed culture. During the operation, temperature was gradually increased from 35 °C to 55 °C, with the highest performance attained at 50 °C. At 50 °C and hydraulic retention time (HRT) of 1.0 d, LA concentration in the broth was 40 kg LA/m(3), corresponding to a yield of 1.6 mol LA/mol hexoseadded. Pyrosequencing results showed that Lactobacillus (97.6% of the total number of sequences) was the predominant species performing LA fermentation of FW. The fermented broth was then centrifuged and LA was extracted from the supernatant by the combined process of nanofiltration and water-splitting electrodialysis. The process could recover highly purified LA by removing 85% of mineral ions such as Na(+), K(+), Mg(2+), and Ca(2+) and 90% of residual carbohydrates. Meanwhile, the solid residue remained after centrifugation was further fermented to biogas by AD. At HRT 40 d (organic loading rate of 7 kg COD/m(3)/d), the highest volumetric biogas production rate of 3.5 m(3)/m(3)/d was achieved with a CH4 yield of 0.25 m(3) CH4/kg COD. The mass flow showed that 47 kg of LA and 54 m(3) of biogas could be recovered by the developed process from 1 ton of FW with COD removal efficiency of 70%. These products have a higher economic value 60 USD/ton FW compared to that of conventional AD (27 USD/ton FW).

  13. Value of estimating methylamalonic acid excretion in anaemia.

    PubMed

    Green, A E; Pegrum, G D

    1968-09-01

    A rapid technique suitable for routine pathology laboratories has been used to estimate methylmalonic acid excretion in a 24-hour urine collection following a 10g. valine load. Levels above 40 mg./24 hours were found only in patients with vitamin B(12) deficiency. Patients with pernicious anaemia treated more than 24 hours before urine collection and patients with other types of anaemia had methylmalonic acid levels below 25 mg./24 hours.This method of demonstrating vitamin B(12) deficiency can be applied rapidly in debilitated patients so that specific treatment can be instituted within 36 hours of admission.

  14. Production of Value-added Products by Lactic Acid Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lactic acid bacteria (LAB) are a group of facultative anaerobic, catalase negative, nonmotile and nonsporeforming–Gram positive bacteria. Most LAB utilize high energy C sources including monomer sugars to produce energy to maintain cellular structure and function. This anaerobic fermentation proce...

  15. [Reference values of folic acid for the Venezuelan population].

    PubMed

    García-Casal, María Nieves; Carias, Diamela; Soto de Sanabria, Ingrid; Victoria López, Ana

    2013-12-01

    The review on folic acid requirements for Venezuela comprise the definitions adopted worldwide known as Dietary Reference Intakes (DRIs) that include Recommended Dietary Allowance (RDA), Estimated Average Requirement (EAR), Adequate Intake (AI) and Tolerable Upper Intake Levels (UL). In contrast with the situation in some countries, that required adjustments in fortification policies in order to avoid excessive folic acid consumption, in Venezuela several studies show an elevated prevalence of deficiency. National evidence at this point is insufficient to establish the recommendation, and as in the 2000 review of the Venezuelan RDAs, the actual recommendations are based on the reported for the United States population. The Recommended Dietary Allowances for Venezuela are 65-80 microg/day for infants less than 1 year old, 150-300 microg/day for children and 400 microg/day for adolescents and adults, increasing to 600 microg/day during pregnancy and to 500 microg/day during lactation. The estimated average requirement is 120-250 microg/day for children, 330 microg/day for adolescents, 320 microg/day for adults, 520 microg/day for pregnancy and 450 microg/day during lactation. The tolerable upper intake levels for folic acid are 300-600 microg/day for children, 800 microg/day for adolescents and 1000 microg/day for adults. During pregnancy and lactation the UL is 800 microg/day for pregnant and lactating women between 14 and 18 years of age, and 1000 microg/day for older pregnant and lactating women. The continuous evaluation of folic acid status is important to design adequate and efficient policies to control both, the deficiency and the excess of folic acid consumption.

  16. A RSK(y) relationship with promiscuous PKA.

    PubMed

    Houslay, Miles D

    2006-08-22

    Where, when, and with "whom" do molecules interact? Such relations in space and time are key concepts that currently engage investigators of cellular signaling processes. The notion of compartmentalized signaling grew out of studies of adenosine 3',5'-monophosphate (cAMP) signaling processes, and this area continues to generate exciting new paradigms. Distinct clouds of cAMP are formed and shaped within cells by tethered cAMP phosphodiesterases (PDEs). AKAPosomes, formed from distinct subpopulations of cAMP-dependent protein kinase (PKA) tethered to anchoring proteins (AKAPs) together with specific substrate molecules, interpret these gradients to generate individualized responses. PKA activity is also regulated by the interaction of other proteins with the regulatory (R) or catalytic (C) subunits of PKA, and a mechanism has been uncovered in which ribosomal S6 kinase (RSK1) interacts with either PKA subunit, depending on whether RSK1 has been phosphorylated and activated by extracellular signal-regulated kinase (ERK). Thus, inactive RSK1 binds the RI subunit of PKA to sensitize it to activation, whereas activated RSK1 binds the C subunit to desensitize PKA to cAMP activation. Cross-talk between the key cAMP and ERK signaling pathways provides a mechanism that, along with distinct mechanisms of both positive and negative attenuation provided by Raf and PDE4 isoforms, can be tailored on a cell type-specific basis.

  17. Anchored PKA as a gatekeeper for gap junctions

    PubMed Central

    Pidoux, Guillaume; Taskén, Kjetil

    2015-01-01

    Anchored protein kinase A (PKA) bound to A Kinase Anchoring Protein (AKAP) mediates effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP-PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin proteins that provide means of communication and transfer of small molecules. While the PKA signaling is known to promote human trophoblast cell fusion, the gap junction communication through connexin 43 (Cx43) is a prerequisite for this process. We recently demonstrated that trophoblast fusion is regulated by ezrin, a known AKAP, which binds to Cx43 and delivers PKA in the vicinity gap junctions. We found that disruption of the ezrin-Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication and subsequently cell fusion. We propose that the PKA-ezrin-Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types. PMID:26478781

  18. Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

    PubMed

    Bombarda, E; Morellet, N; Cherradi, H; Spiess, B; Bouaziz, S; Grell, E; Roques, B P; Mély, Y

    2001-07-13

    The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant

  19. Controlling the pKa of the bacteriorhodopsin Schiff base by use of artificial retinal analogues

    SciTech Connect

    Sheves, M.; Albeck, A.; Friedman, N.; Ottolenghi, M.

    1986-05-01

    Artificial bacteriorhodopsin pigments based on synthetic retinal analogues carrying an electron-withdrawing CF3 substituent group were prepared. The effects of CF3 on the spectra, photocycles, and Schiff base pKa values of the pigments were analyzed. A reduction of 5 units in the pKa of the Schiff base is observed when the CF3 substituent is located at the C-13 polyene position, in the vicinity of the protonated Schiff base nitrogen. The results lead to the unambiguous characterization of the (direct) titration of the Schiff base in bacteriorhodopsin and to the conclusion that the deprotonation rate of the Schiff base during the photocycle (i.e., the generation of the M412 intermediate) is determined by a structural change in the protein.

  20. pH indicator titration: a novel fast pKa determination method.

    PubMed

    Kong, Xiaole; Zhou, Tao; Liu, Zudong; Hider, Robert C

    2007-10-01

    This study describes a fast spectrophotometic titration method for apparent ionization constant (pKa) determination. In this method, a Universal pH indicator is utilized instead of the conventional pH electrode. An autoburette is set to add HCl at a constant rate to a vigorously stirred 1 cm UV cuvette which contains sample and indicator solution. A spectrophotometer continuously records the spectra. Acquired spectral data are processed by calculating the pH from the indicator spectra in the visible region and extracting sample spectra from the UV region. Five compounds possessing pKa values in the range 2-10 were investigated. These results differed from measurements by conventional spectrophotometric titration by +/-0.05 to +/-0.10 log unit.

  1. Surface acidity of quartz: understanding the crystallographic control.

    PubMed

    Liu, Xiandong; Cheng, Jun; Lu, Xiancai; Wang, Rucheng

    2014-12-28

    We report a first principles molecular dynamics (FPMD) study of surface acid chemistry of the two growth surfaces of quartz, (101̄0) (including Alpha and Beta terminations) and (101̄1) facets. The interfacial hydration structures are characterized in detail and the intrinsic pKas of surface silanols are evaluated using the FPMD based vertical energy gap method. The calculated acidity constants reveal that every surface termination shows a bimodal acid-base behavior. It is found that all doubly-protonated forms (i.e. SiOH2) on the three terminations have pKas lower than -2.5, implying that the silanols hardly get protonated in a common pH range. The pKas of surface silanols can be divided into 3 groups. The most acidic silanol is the donor SiOH on the (101̄0)-beta surface (pKa = 4.8), the medium includes the germinal silanol on (101̄0)-alpha and the outer silanol on (101̄1) (pKa = 8.5-9.3) and the least acidic are inner silanols on the (101̄1)-facet, acceptor SiOH on (101̄0)-beta, and the secondly-deprotonated OH (i.e. Si(O-)(OH)) on (101̄0)-alpha (pKa > 11.0). With the pKa values, we discuss the implication for understanding metal cations complexing on quartz surfaces.

  2. β-(1-Azulenyl)-L-alanine--a functional probe for determination of pKa of histidine residues.

    PubMed

    Gosavi, Pallavi M; Moroz, Yurii S; Korendovych, Ivan V

    2015-03-28

    β-(1-Azulenyl)-L-alanine (AzAla) can be incorporated into the influenza A virus M2 proton channel. AzAla's sensitivity to the protonation state of the nearby histidines and the lack of environmental fluorescence dependence allow for direct and straightforward determination of histidine pKa values in ion channels.

  3. Determination of acid-base dissociation constants of azahelicenes by capillary zone electrophoresis.

    PubMed

    Ehala, Sille; Mísek, Jirí; Stará, Irena G; Starý, Ivo; Kasicka, Václav

    2008-08-01

    CZE was employed to determine acid-base dissociation constants (pK(a)) of ionogenic groups of azahelicenes in methanol (MeOH). Azahelicenes are unique 3-D aromatic systems, which consist of ortho-fused benzene/pyridine units and exhibit helical chirality. The pK(a) values of pyridinium groups of the studied azahelicenes were determined from the dependence of their effective electrophoretic mobility on pH by a nonlinear regression analysis. The effective mobilities of azahelicenes were determined by CZE at pH range between 2.1 and 10.5. Thermodynamic pK(a) values of monobasic 1-aza[6]helicene and 2-aza[6]helicene in MeOH were determined to be 4.94 +/- 0.05 and 5.68 +/- 0.05, respectively, and pK(a) values of dibasic 1,14-diaza[5]helicene were found to be equal to 7.56 +/- 0.38 and 8.85 +/- 0.26. From these values, the aqueous pK(a) of these compounds was estimated.

  4. Absorption Spectroscopy Study of Acid-Base and Metal-Binding Properties of Flavanones

    NASA Astrophysics Data System (ADS)

    Shubina, V. S.; Shatalina, Yu. V.

    2013-11-01

    We have used absorption spectroscopy to study the acid-base and metal-binding properties of two structurally similar flavanones: taxifolin and naringenin. We have determined the acid dissociation constants for taxifolin (pKa1 = 7.10 ± 0.05, pKa2 = 8.60 ± 0.09, pKa3 = 8.59 ± 0.19, pKa4 = 11.82 ± 0.36) and naringenin (pKa1 = 7.05 ± 0.05, pKa2 = 8.85 ± 0.09, pKa3 = 12.01 ± 0.38). The appearance of new absorption bands in the visible wavelength region let us determine the stoichiometric composition of the iron (II) complexes of the flavanones. We show that at pH 5, in solution there is a mixture of complexes between taxifolin and iron (II) ions in stoichiometric ratio 2:1 and 1:2, while at pH 7.4 and pH 9, we detect a 1:1 taxifolin:Fe(II) complex. We established that at these pH values, naringenin forms a 2:1 complex with iron (II) ions. We propose structures for the complexes formed. Comprehensive study of the acid-base properties and the metal-binding capability of the two structurally similar flavanones let us determine the structure-properties relation and the conditions under which antioxidant activity of the polyphenols appears, via chelation of variable-valence metal ions.

  5. Determination of the acid dissociation constant of bromocresol green and cresol red in water/AOT/isooctane reverse micelles by multiple linear regression and extended principal component analysis.

    PubMed

    Caselli, Maurizio; Mangone, Annarosa; Paolillo, Paola; Traini, Angela

    2002-01-01

    The pKa of 3',3",5',5"tetrabromo-m-cresolsulfonephtalein (Bromocresol Green) and o-cresolsulphonephtalein (Cresol Red) was spectrophotometrically measured in a water/AOT/isooctane microemulsion in the presence of a series of buffers carrying different charges at different water/surfactant ratios. Extended Principal Component Analysis was used for a precise determination of the apparent pKa and of the spectra of the acid and base forms of the dye. The apparent pKa of dyes in water-in-oil microemulsions depends on the charge of the acid and base forms of the buffers present in the water pool. Combination with multiple linear regression increases the precision. Results are discussed taking into account the profile of the electrostatic potential in the water pool and the possible partition of the indicator between the aqueous core and the surfactant. The pKa corrected for these effects are independent of w0 and are close to the value of the pKa in bulk water. On the basis of a tentative hypothesis it is possible to calculate the true pKa of the buffer in the pool.

  6. Application of Multiple Linear Regression and Extended Principal-Component Analysis to Determination of the Acid Dissociation Constant of 7-Hydroxycoumarin in Water/AOT/Isooctane Reverse Micelles.

    PubMed

    Caselli; Daniele; Mangone; Paolillo

    2000-01-15

    The apparent pK(a) of dyes in water-in-oil microemulsions depends on the charge of the acid and base forms of the buffers present in the water pool. Extended principal-component analysis allows the precise determination of the apparent pK(a) and of the spectra of the acid and base forms of the dye. Combination with multiple linear regression increases the precision. The pK(a) of 7-hydroxycoumarin (umbelliferone) was spectrophotometrically measured in a water/AOT/isooctane microemulsion in the presence of a series of buffers carrying different charges at various different water/surfactant ratios. The spectra of the acid and base forms of the dye in the microemulsion are very similar to those in bulk water in the presence of Tris and ammonia. The presence of carbonate changes somewhat the spectrum of the acid form. Results are discussed taking into account the profile of the electrostatic potential drop in the water pool and the possible partition of umbelliferone between the aqueous core and the surfactant. The pK(a) values corrected for these effects are independent of w(0) and are close to the value of the pK(a) in bulk water. Copyright 2000 Academic Press.

  7. Thiazolium C(2)-proton exchange: structure-reactivity correlations and the pKa of thiamin C(2)-H revisited.

    PubMed

    Washabaugh, M W; Jencks, W P

    1988-07-12

    Rate constants for C(2)-proton exchange from thiamin, N(1')-methylthiamin, and several 3-substituted-4-methylthiazolium ions catalyzed by D2O and deuterioxide ion were determined by 1H NMR at 30 degrees C and ionic strength 2.0 M. Values of pKa for the thiazolium ions, including thiamin itself, were found to be in the range pKa = 17-19; the pKa values for N(1')-protonated thiamin and free thiamin C(2)-H in H2O are 17.7 and 18.0, respectively. The pKa value for N(1')-protonated thiamin was calculated from the observed rate constant for the pD-independent reaction with D2O after correction for a secondary solvent deuterium isotope effect of kH2O/kD2O = 2.6. The pKa value for free thiamin was calculated from the rate constant for catalysis by OD- after correction by a factor of 3.3 = 8/2.4 for an 8-fold negative deviation of kOD from the Brønsted plot of slope 1.0 for general base catalysis and a secondary solvent isotope effect of kOD/kOH = 2.4. Values of k-a = 2 X 10(10) and 3 X 10(9) M-1 s-1 were assumed for diffusion-controlled protonation of the C(2) ylide in the reverse direction by H3O+ and H2O, respectively. The Hammett rho I value for the exchange reaction catalyzed by deuterioxide ion or D2O is 8.4 +/- 0.2. There is no positive deviation of the rate constants for free or N(1')-substituted thiamin analogues in either Hammett correlation. This shows that the aminopyrimidinyl group does not provide significant intramolecular catalysis of nonenzymic C(2)-proton removal in the coenzyme.

  8. Determination of the acid dissociation constant of the biosurfactant monorhamnolipid in aqueous solution by potentiometric and spectroscopic methods.

    PubMed

    Lebrón-Paler, Ariel; Pemberton, Jeanne E; Becker, Bridget A; Otto, William H; Larive, Cynthia K; Maier, Raina M

    2006-11-15

    The acid dissociation constant in water for a monorhamnolipid mixture extracted from Pseudomonas aeruginosa ATCC 9027 has been determined using potentiometry and two spectroscopic approaches at concentrations below and above the critical micelle concentration (cmc). Potentiometric titrations resulted in pKa values ranging from 4.28 +/- 0.16 to 5.50 +/- 0.06 depending on concentration. 1H NMR spectrochemical titrations at concentrations below the cmc revealed a pKa value of 4.39 +/- 0.06. ATR-FT-IR spectrochemical titrations on solutions well above the cmc gave a pKa value of 4.84 +/- 0.05. The value of 4.28 for the free rhamnolipid molecule for concentrations below the cmc differs markedly from that reported previously. However, the pKa of 5.50 for surface-adsorbed and solution aggregates correlates closely to that previously reported. Differences in these pKa values are rationalized in terms of the pH- and concentration-dependent aggregation behavior of rhamnolipids in aqueous solution.

  9. Best of both worlds: combining pharma data and state of the art modeling technology to improve in Silico pKa prediction.

    PubMed

    Fraczkiewicz, Robert; Lobell, Mario; Göller, Andreas H; Krenz, Ursula; Schoenneis, Rolf; Clark, Robert D; Hillisch, Alexander

    2015-02-23

    In a unique collaboration between a software company and a pharmaceutical company, we were able to develop a new in silico pKa prediction tool with outstanding prediction quality. An existing pKa prediction method from Simulations Plus based on artificial neural network ensembles (ANNE), microstates analysis, and literature data was retrained with a large homogeneous data set of drug-like molecules from Bayer. The new model was thus built with curated sets of ∼14,000 literature pKa values (∼11,000 compounds, representing literature chemical space) and ∼19,500 pKa values experimentally determined at Bayer Pharma (∼16,000 compounds, representing industry chemical space). Model validation was performed with several test sets consisting of a total of ∼31,000 new pKa values measured at Bayer. For the largest and most difficult test set with >16,000 pKa values that were not used for training, the original model achieved a mean absolute error (MAE) of 0.72, root-mean-square error (RMSE) of 0.94, and squared correlation coefficient (R(2)) of 0.87. The new model achieves significantly improved prediction statistics, with MAE = 0.50, RMSE = 0.67, and R(2) = 0.93. It is commercially available as part of the Simulations Plus ADMET Predictor release 7.0. Good predictions are only of value when delivered effectively to those who can use them. The new pKa prediction model has been integrated into Pipeline Pilot and the PharmacophorInformatics (PIx) platform used by scientists at Bayer Pharma. Different output formats allow customized application by medicinal chemists, physical chemists, and computational chemists.

  10. Effect of temperature and solvent composition on acid dissociation equilibria, I: Sequenced (s)(s)pKa determination of compounds commonly used as buffers in high performance liquid chromatography coupled to mass spectroscopy detection.

    PubMed

    Padró, Juan M; Acquaviva, Agustín; Tascon, Marcos; Gagliardi, Leonardo G; Castells, Cecilia B

    2012-05-01

    A new automated and rapid potentiometric method for determining the effect of organic-solvent composition on pK(a) has been developed. It is based on the measurements of pH values of buffer solutions of variable solvent compositions using a combined glass electrode. Additions of small volumes of one precisely thermostated solution into another, both containing exactly the same analytical concentrations of the buffer components, can produce continuous changes in the solvent composition. Two sequences of potential measurements, one of increasing and the other of decreasing solvent content, are sufficient to obtain the pK(a) values of the acidic compound within the complete solvent-composition range in about 2h. The experimental design, procedures, and calculations needed to convert the measured pH into the thermodynamic pK(a) values are thoroughly discussed. This rapid and automated method allows the systematic study of the effect of solvent compositions and temperatures on the pK(a). It has been applied to study the dissociation constants of two monoprotic acids: formic acid and triethylamine:HCl in acetonitrile/water mixtures within the range from 0 to 90% (v/v) at temperatures between 20°C and 60°C. These volatile compounds are frequently used to control the pH of the mobile phase in HPLC, especially in methods coupled to mass-spectrometry detection. The obtained pK(a) values are in excellent agreement with those previously reported. The results were fitted to empirical functions between pK(a) and temperature and composition. These equations, which can be used to estimate the pK(a) of these substances at any composition and temperature, would be highly useful in practical work during chromatographic method development.

  11. Role of PKA in regulating mitochondrial function and neuronal development: implications to neurodegenerative diseases

    PubMed Central

    Dagda, Ruben K.; Banerjee, Tania Das

    2015-01-01

    In neurons, enhanced PKA signaling elevates synaptic plasticity, promotes neuronal development, and increases dopamine synthesis. On the other hand, a decline in PKA signaling contributes to the etiology of several brain degenerative diseases including Alzheimer’s disease and Parkinson’s disease suggesting that PKA predominantly plays a neuroprotective role. A-kinase anchoring proteins (AKAP) are large multi-domain scaffold proteins that target PKA and other signaling molecules to distinct subcellular sites to strategically localize PKA signaling at dendrites, dendritic spines, cytosol, and axons. PKA can be recruited to outer mitochondrial membrane by associating with three different AKAPs to regulate mitochondrial dynamics, structure, mitochondrial respiration, trafficking, dendrite morphology, and neuronal survival. In this review, we survey the myriad of essential neuronal functions modulated by PKA but place a special emphasis on mitochondrially-localized PKA. Finally, we offer an updated overview of how loss of PKA signaling contributes to the etiology of several brain degenerative diseases. PMID:25741943

  12. Bimodal antagonism of PKA signalling by ARHGAP36

    PubMed Central

    Eccles, Rebecca L.; Czajkowski, Maciej T.; Barth, Carolin; Müller, Paul Markus; McShane, Erik; Grunwald, Stephan; Beaudette, Patrick; Mecklenburg, Nora; Volkmer, Rudolf; Zühlke, Kerstin; Dittmar, Gunnar; Selbach, Matthias; Hammes, Annette; Daumke, Oliver; Klussmann, Enno; Urbé, Sylvie; Rocks, Oliver

    2016-01-01

    Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease. PMID:27713425

  13. Analyses of the involvement of PKA regulation mechanism in meiotic incompetence of porcine growing oocytes.

    PubMed

    Nishimura, Takanori; Fujii, Wataru; Kano, Kiyoshi; Sugiura, Koji; Naito, Kunihiko

    2012-09-01

    Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. In the present study, we cloned cDNAs of cAMP-dependent protein-kinase (PKA) subunits from porcine oocytes and analyzed the involvement of the PKA regulation mechanism in the meiotic incompetence of GOs at the molecular level. We found a cAMP-independent high PKA activity in GOs throughout the in vitro culture using a porcine PKA assay system we established, and inhibition of the activity by injection of the antisense RNA of the PKA catalytic subunit (PKA-C) induced meiotic resumption in GOs. Then we examined the possibility that the amount of the PKA regulatory subunit (PKA-R), which can bind and inhibit PKA-C, was insufficient to suppress PKA activity in GOs because of the overexpression of two PKA-Rs, PRKAR1A and PRKAR2A. We found that neither of them affected PKA activity and induced meiotic resumption in GO although PRKAR2A could inhibit PKA activity and induce meiosis in cAMP-treated full-grown oocytes (FGOs). Finally, we analyzed the subcellular localization of PKA subunits and found that all the subunits were localized in the cytoplasm during meiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A, entered into the nucleus just before meiotic resumption in FGOs, whereas all of them remained in the cytoplasm in GOs throughout the culture period. Our findings suggest that the continuous high PKA activity is a primary cause of the meiotic incompetence of porcine GOs and that this PKA activity is not simply caused by an insufficient expression level of PKA-R, but can be attributed to more complex spatial-temporal regulation mechanisms. PMID:22674394

  14. Panax red ginseng extract regulates energy expenditures by modulating PKA dependent lipid mobilization in adipose tissue.

    PubMed

    Cho, Hae-Mi; Kang, Young-Ho; Yoo, Hanju; Yoon, Seung-Yong; Kang, Sang-Wook; Chang, Eun-Ju; Song, Youngsup

    2014-05-16

    Regulation of balance between lipid accumulation and energy consumption is a critical step for the maintenance of energy homeostasis. Here, we show that Panax red ginseng extract treatments increased energy expenditures and prevented mice from diet induced obesity. Panax red ginseng extracts strongly activated Hormone Specific Lipase (HSL) via Protein Kinase A (PKA). Since activation of HSL induces lipolysis in WAT and fatty acid oxidation in brown adipose tissue (BAT), these results suggest that Panax red ginseng extracts reduce HFD induced obesity by regulating lipid mobilization.

  15. Polysaccharide Nanoparticles for Efficient siRNA Targeting in Cancer Cells by Supramolecular pKa Shift

    PubMed Central

    Zhang, Ying-Ming; Yang, Yang; Zhang, Yu-Hui; Liu, Yu

    2016-01-01

    Biomacromolecular pKa shifting is considered as one of the most ubiquitous processes in biochemical events, e.g., the enzyme-catalyzed reaction and protein conformational stabilization. In this paper, we report on the construction of biocompatible polysaccharide nanoparticle with targeting ability and lower toxicity by supramolecular pKa shift strategy. This was realized through a ternary assembly constructed by the dual host‒guest interactions of an adamantane-bis(diamine) conjugate (ADA) with cucurbit[6]uril (CB[6]) and a polysaccharide. The potential application of such biocompatible nanostructure was further implemented by the selective transportation of small interfering RNA (siRNA) in a controlled manner. It is demonstrated that the strong encapsulation of the ADA’s diammonium tail by CB[6] not only reduced the cytotoxicity of the nano-scaled vehicle but also dramatically enhanced cation density through an obvious positive macrocycle-induced pKa shift, which eventually facilitated the subsequent siRNA binding. With a targeted polysaccharide shell containing a cyclodextrin‒hyaluronic acid conjugate, macrocycle-incorporated siRNA polyplexes were specifically delivered into malignant human prostate PC-3 cells. The supramolecular polysaccharide nanoparticles, the formation of which was enabled and promoted by the complexation-assisted pKa shift, may be used as a versatile tool for controlled capture and release of biofunctional substrates. PMID:27363811

  16. Polysaccharide Nanoparticles for Efficient siRNA Targeting in Cancer Cells by Supramolecular pKa Shift

    NASA Astrophysics Data System (ADS)

    Zhang, Ying-Ming; Yang, Yang; Zhang, Yu-Hui; Liu, Yu

    2016-07-01

    Biomacromolecular pKa shifting is considered as one of the most ubiquitous processes in biochemical events, e.g., the enzyme-catalyzed reaction and protein conformational stabilization. In this paper, we report on the construction of biocompatible polysaccharide nanoparticle with targeting ability and lower toxicity by supramolecular pKa shift strategy. This was realized through a ternary assembly constructed by the dual host‒guest interactions of an adamantane-bis(diamine) conjugate (ADA) with cucurbit[6]uril (CB[6]) and a polysaccharide. The potential application of such biocompatible nanostructure was further implemented by the selective transportation of small interfering RNA (siRNA) in a controlled manner. It is demonstrated that the strong encapsulation of the ADA’s diammonium tail by CB[6] not only reduced the cytotoxicity of the nano-scaled vehicle but also dramatically enhanced cation density through an obvious positive macrocycle-induced pKa shift, which eventually facilitated the subsequent siRNA binding. With a targeted polysaccharide shell containing a cyclodextrin‒hyaluronic acid conjugate, macrocycle-incorporated siRNA polyplexes were specifically delivered into malignant human prostate PC-3 cells. The supramolecular polysaccharide nanoparticles, the formation of which was enabled and promoted by the complexation-assisted pKa shift, may be used as a versatile tool for controlled capture and release of biofunctional substrates.

  17. Determination of proton affinities and acidity constants of sugars.

    PubMed

    Feng, Shuting; Bagia, Christina; Mpourmpakis, Giannis

    2013-06-20

    Proton transfer reactions play a key role in the conversion of biomass derived sugars to chemicals. In this study, we employ high level ab initio theoretical methods, in tandem with solvation effects to calculate the proton affinities (PA) and acidity constants (pKa) of various d-glucose and d-fructose tautomers (protonation-deprotonation processes). In addition, we compare the theoretically derived pH values of sugar solutions against experimentally measured pH values in our lab. Our results demonstrate that the protonation of any of the O atoms of the sugars is thermodynamically preferred without any significant variation in the PA values. Intramolecular hydrogen transfers, dehydration reactions, and ring-opening processes were observed, resulting from the protonation of specific hydroxyl groups on the sugars. Regarding the deprotonation processes (pKa), we found that the sugars' anomeric hydroxyls exhibit the highest acidity. The theoretically calculated pH values of sugar solutions are in excellent agreement with experimental pH measurements at low sugar concentrations. At higher sugar concentrations the calculations predict less acidic solutions than the experiments. In this case, we expect the sugars to act as solvents increasing the proton solvation energy and the acidity of the solutions. We demonstrated through linear relationships that the pKa values are correlated with the relative stability of the conjugate bases. The latter is related to hydrogen bonding and polarization of the C-O(-) bond. A plausible explanation for the good performance of the direct method in calculating the pKa values of sugars can be the presence of intramolecular hydrogen bonds on the conjugate base. Both theory and experiments manifest that fructose is a stronger acid than glucose, which is of significant importance in self-catalyzed biomass-relevant dehydration reactions. PMID:23706015

  18. [Progress in engineering Escherichia coli for production of high-value added organic acids and alcohols].

    PubMed

    Wang, Jiming; Liu, Wei; Xu, Xin; Zhang, Haibo; Xian, Mo

    2013-10-01

    Confronted with the gradual exhaustion of the earth's fossil energy resources and the grimmer environmental deterioration, the bio-based process to produce high-value added platform chemicals from renewable biomass is attracting growing interest. Escherichia coli has been chosen as a workhouse for the production of many valuable chemicals due to various advantages, such as clear genetic background, convenient to be genetically modified and good growth properties with low nutrient requirements. Rational strain development of E. coli achieved by metabolic engineering strategies has provided new processes for efficiently biotechnological production of various high-value chemical building blocks. This review focuses on recent progresses in metabolic engineering of E. coli that lead to efficient recombinant biocatalysts for production of high-value organic acids such as succinic acid, 3-hydroxypropanoic acid and glucaric acid as well as alcohols like glycerol and xylitol. Besides, this review also discusses several other platform chemicals, including 2,5-furan dicarboxylic acid, aspartic acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxy-gamma-butyrolactone and sorbitol, which have not been produced by E. coli until now. PMID:24432652

  19. Direct production of biodiesel from high-acid value Jatropha oil with solid acid catalyst derived from lignin

    PubMed Central

    2011-01-01

    Background Solid acid catalyst was prepared from Kraft lignin by chemical activation with phosphoric acid, pyrolysis and sulfuric acid. This catalyst had high acid density as characterized by scanning electron microscope (SEM), energy-dispersive x-ray spectrometry (EDX) and Brunauer, Emmett, and Teller (BET) method analyses. It was further used to catalyze the esterification of oleic acid and one-step conversion of non-pretreated Jatropha oil to biodiesel. The effects of catalyst loading, reaction temperature and oil-to-methanol molar ratio, on the catalytic activity of the esterification were investigated. Results The highest catalytic activity was achieved with a 96.1% esterification rate, and the catalyst can be reused three times with little deactivation under optimized conditions. Biodiesel production from Jatropha oil was studied under such conditions. It was found that 96.3% biodiesel yield from non-pretreated Jatropha oil with high-acid value (12.7 mg KOH/g) could be achieved. Conclusions The catalyst can be easily separated for reuse. This single-step process could be a potential route for biodiesel production from high-acid value oil by simplifying the procedure and reducing costs. PMID:22145867

  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. Assessment of the effects of iodine value on fatty acid digestibility, feed intake, and milk production.

    PubMed

    Firkins, J L; Eastridge, M L

    1994-08-01

    Data were pooled from 11 studies evaluating supplemental fat sources differing primarily in degree of saturation (tallow, animal-vegetable fat, vegetable oil, and hydrogenated fats). Data were standardized as proportions of the respective controls to reduce variation among individual studies and were subjected to stepwise multiple regression against the iodine value of fats, the percentage increases of total fatty acids in diets above the respective controls, or the ratio of total C16 to C18 fatty acids in fats (only for hydrogenated fats). Increased iodine value (increasing unsaturation) of fats increased apparent fatty acid digestibility, especially as iodine value increased from 11 to 27. For partially hydrogenated fat sources, as the ratio of C16 to C18 fatty acids increased, digestibility also increased, especially with increasing iodine value (positive interaction). Beneficial effects of higher C16:C18 ratio were reduced as amount of added fat increased (negative interaction). Dry matter intake and FCM production decreased as iodine value increased, perhaps because of inhibition of fiber digestion or metabolic regulation of DMI. Milk protein percentage depression averaged .2 percentage units for most fats. However, as partially hydrogenated fat sources became more saturated, milk protein depression appeared to be less evident; increased ratio of C16:C18 of fatty acids appeared to increase milk protein percentage. Despite the lower apparent digestibility of fatty acids of hydrogenated fats, increased milk production and percentages of fat and protein appeared to make them more economical than more unsaturated fats.

  2. PKA isoforms coordinate mRNA fate during nutrient starvation

    PubMed Central

    Tudisca, Vanesa; Simpson, Clare; Castelli, Lydia; Lui, Jennifer; Hoyle, Nathaniel; Moreno, Silvia; Ashe, Mark; Portela, Paula

    2012-01-01

    Summary A variety of stress conditions induce mRNA and protein aggregation into mRNA silencing foci, but the signalling pathways mediating these responses are still elusive. Previously we demonstrated that PKA catalytic isoforms Tpk2 and Tpk3 localise with processing and stress bodies in Saccharomyces cerevisiae. Here, we show that Tpk2 and Tpk3 are associated with translation initiation factors Pab1 and Rps3 in exponentially growing cells. Glucose starvation promotes the loss of interaction between Tpk and initiation factors followed by their accumulation into processing bodies. Analysis of mutants of the individual PKA isoform genes has revealed that the TPK3 or TPK2 deletion affects the capacity of the cells to form granules and arrest translation properly in response to glucose starvation or stationary phase. Moreover, we demonstrate that PKA controls Rpg1 and eIF4G1 protein abundance, possibly controlling cap-dependent translation. Taken together, our data suggest that the PKA pathway coordinates multiple stages in the fate of mRNAs in association with nutritional environment and growth status of the cell. PMID:22899713

  3. Determination of acid-base dissociation constants of amino- and guanidinopurine nucleotide analogs and related compounds by capillary zone electrophoresis.

    PubMed

    Solínová, Veronika; Kasicka, Václav; Koval, Dusan; Cesnek, Michal; Holý, Antonín

    2006-03-01

    CZE has been applied for determination of acid-base dissociation constants (pKa) of ionogenic groups of newly synthesized amino- and (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonate, acyclic nucleoside phosphonate diesters and other related compounds. These compounds bear characteristic pharmacophores contained in various important biologically active substances, such as cytostatics and antivirals. The pKa values of ionogenic groups of the above compounds were determined by nonlinear regression analysis of the experimentally measured pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by CZE performed in series of BGEs in a broad pH range (3.50-11.25), at constant ionic strength (25 mM) and temperature (25 degrees C). pKa values were determined for the protonated guanidinyl group in (amino)guanidino 9-alkylpurines and in (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonates and acyclic nucleoside phosphonate diesters, for phosphonic acid to the second dissociation degree (-2) in acyclic nucleoside phosphonates of amino and (amino)guanidino 9-alkylpurines, and for protonated nitrogen in position 1 (N1) of purine moiety in acyclic nucleoside phosphonates of amino 9-alkylpurines. Thermodynamic pKa of protonated guanidinyl group was estimated to be in the range of 7.75-10.32, pKa of phosphonic acid to the second dissociation degree achieved values of 6.64-7.46, and pKa of protonated nitrogen in position 1 of purine was in the range of 4.13-4.89, depending on the structure of the analyzed compounds.

  4. Effect of interior loop length on the thermal stability and pK(a) of i-motif DNA.

    PubMed

    Reilly, Samantha M; Morgan, Rhianna K; Brooks, Tracy A; Wadkins, Randy M

    2015-02-17

    The four-stranded i-motif (iM) conformation of cytosine-rich DNA is important in a wide variety of biochemical systems ranging from its use in nanomaterials to a potential role in oncogene regulation. An iM is stabilized by acidic pH that allows hemiprotonated cytidines to form a C·C(+) base pair. Fundamental studies that aim to understand how the lengths of loops connecting the protonated C·C(+) pairs affect intramolecular iM physical properties are described here. We characterized both the thermal stability and the pK(a) of intramolecular iMs with differing loop lengths, in both dilute solutions and solutions containing molecular crowding agents. Our results showed that intramolecular iMs with longer central loops form at pHs and temperatures higher than those of iMs with longer outer loops. Our studies also showed that increases in thermal stability of iMs when molecular crowding agents are present are dependent on the loop that is lengthened. However, the increase in pK(a) for iMs when molecular crowding agents are present is insensitive to loop length. Importantly, we also determined the proton activity of solutions containing high concentrations of molecular crowding agents to ascertain whether the increase in pK(a) of an iM is caused by alteration of this activity in buffered solutions. We determined that crowding agents alone increase the apparent pK(a) of a number of small molecules as well as iMs but that increases to iM pK(a) were greater than that expected from a shift in proton activity.

  5. Effect of high-energy electron irradiation of chicken meat on thiobarbituric acid values, shear values, odor, and cooked yield

    SciTech Connect

    Heath, J.L.; Owens, S.L.; Tesch, S.; Hannah, K.W. )

    1990-02-01

    Experiments were conducted to determine whether electron-beam irradiation would affect shear values, yield, odor, and thiobarbituric acid (TBA) values of chicken tissues. Broiler breasts (pectoralis superficialis) and whole thighs were irradiated with an electron-beam accelerator at levels to produce adsorbed doses of 100, 200, and 300 krads on the surface of the sample. The thigh samples were stored for 2, 4, and 8 days before testing for TBA values. The depth to which the radiation had penetrated the pectoralis superficialis muscle was also determined. Radiation penetrated 22 mm into slices of pectoralis superficialis muscle when 100 krad was absorbed by the surface of the tissue. The dose absorbed beneath the tissue surface to a depth of 10 mm was larger than the dose absorbed at the surface. The absorbed dose decreased as the depth of penetration increased. For cooked breast tissue, the shear values and moisture content were not affected by the absorbed radiation. Cooking losses of aged breast tissue were not affected by irradiation, but cooking losses were reduced in breast tissue that had not been aged. Irradiating uncooked thigh and uncooked breast samples produced a characteristic odor that remained after the thighs were cooked but was not detectable after the breast samples were cooked. With two exceptions, no significantly different TBA values were found that could be attributed to irradiation.

  6. Distinct Effects of Sorbic Acid and Acetic Acid on the Electrophysiology and Metabolism of Bacillus subtilis

    PubMed Central

    van Beilen, J. W. A.; Teixeira de Mattos, M. J.; Hellingwerf, K. J.

    2014-01-01

    Sorbic acid and acetic acid are among the weak organic acid preservatives most commonly used to improve the microbiological stability of foods. They have similar pKa values, but sorbic acid is a far more potent preservative. Weak organic acids are most effective at low pH. Under these circumstances, they are assumed to diffuse across the membrane as neutral undissociated acids. We show here that the level of initial intracellular acidification depends on the concentration of undissociated acid and less on the nature of the acid. Recovery of the internal pH depends on the presence of an energy source, but acidification of the cytosol causes a decrease in glucose flux. Furthermore, sorbic acid is a more potent uncoupler of the membrane potential than acetic acid. Together these effects may also slow the rate of ATP synthesis significantly and may thus (partially) explain sorbic acid's effectiveness. PMID:25038097

  7. The effect of manuring on cereal and pulse amino acid δ(15)N values.

    PubMed

    Styring, Amy K; Fraser, Rebecca A; Bogaard, Amy; Evershed, Richard P

    2014-06-01

    Amino acid δ(15)N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown in manured and unmanured soil at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. Manuring was found to result in a consistent (15)N-enrichment of cereal grain amino acid δ(15)N values, indicating that manuring did not affect the metabolic routing of nitrogen (N) into cereal grain amino acids. The increase in cereal grain δ(15)N values with manuring is therefore due to a (15)N-enrichment in the δ(15)N value of assimilated inorganic-N. Greater variation was observed in the (15)N-enrichment of rachis amino acids with manuring, possibly due to enhanced sensitivity to changes in growing conditions and higher turnover of N in rachis cells compared to cereal grains. Total amino acid δ(15)N values of manured and unmanured broad beans and peas were very similar, indicating that the legumes assimilated N2 from the atmosphere rather than N from the soil, since there was no evidence for routing of (15)N-enriched manure N into any of the pulse amino acids. Crop amino acid δ(15)N values thus provide insights into the sources of N assimilated by non N2-fixing and N2-fixing crops grown on manured and unmanured soils, and reveal an effect of manure on N metabolism in different crop species and plant parts.

  8. An NMR and ab initio quantum chemical study of acid-base equilibria for conformationally constrained acidic alpha-amino acids in aqueous solution.

    PubMed

    Nielsen, P A; Jaroszewski, J W; Norrby, P O; Liljefors, T

    2001-03-01

    The protonation states of a series of piperidinedicarboxylic acids (PDAs), which are conformationally constrained acidic alpha-amino acids, have been studied by (13)C NMR titration in water. The resulting data have been correlated with theoretical results obtained by HF/6-31+G calculations using the polarizable continuum model (PCM) for the description of water. The PDAs are highly ionizable and contain one or two possible internal hydrogen bonds. In the present study, we show that the PCM model is able to reproduce the relative stabilities of the different protonation states of the PDAs. Furthermore, our results show that prediction of relative pK(a) values for two different types of ionizable functional groups covering a pK(a) range from 1.6 to 12.1 is possible with a high degree of accuracy.

  9. Estimating the acidity of transition metal hydride and dihydrogen complexes by adding ligand acidity constants.

    PubMed

    Morris, Robert H

    2014-02-01

    A simple equation (pKa(THF) = ∑AL + Ccharge + Cnd + Cd6) can be used to obtain an estimate of the pKa of diamagnetic transition metal hydride and dihydrogen complexes in tetrahydrofuran, and, by use of conversion equations, in other solvents. It involves adding acidity constants AL for each of the ligands in the 5-, 6-, 7-, or 8-coordinate conjugate base complex of the hydride or dihydrogen complex along with a correction for the charge (Ccharge = -15, 0 or 30 for x = +1, 0 or -1 charge, respectively) and the periodic row of the transition metal (Cnd = 0 for 3d or 4d metal, 2 for 5d metal) as well as a correction for d(6) octahedral acids (Cd6 = 6 for d(6) metal ion in the acid, 0 for others) that are not dihydrogen complexes. Constants AL are provided for 13 commonly occurring ligand types; of these, nine neutral ligands are correlated with Lever's electrochemical ligand parameters EL. This method gives good estimates of the over 170 literature pKa values that range from less than zero to 50 with a standard deviation of 3 pKa units for complexes of the metals chromium to nickel, molybdenum, ruthenium to palladium, and tungsten to platinum in the periodic table. This approach allows a quick assessment of the acidity of hydride complexes found in nature (e.g., hydrogenases) and in industry (e.g., catalysis and hydrogen energy applications). The pKa values calculated for acids that have bulky or large bite angle chelating ligands deviate the most from this correlation. The method also provides an estimate of the base strength of the deprotonated form of the complex.

  10. Estimating the acidity of transition metal hydride and dihydrogen complexes by adding ligand acidity constants.

    PubMed

    Morris, Robert H

    2014-02-01

    A simple equation (pKa(THF) = ∑AL + Ccharge + Cnd + Cd6) can be used to obtain an estimate of the pKa of diamagnetic transition metal hydride and dihydrogen complexes in tetrahydrofuran, and, by use of conversion equations, in other solvents. It involves adding acidity constants AL for each of the ligands in the 5-, 6-, 7-, or 8-coordinate conjugate base complex of the hydride or dihydrogen complex along with a correction for the charge (Ccharge = -15, 0 or 30 for x = +1, 0 or -1 charge, respectively) and the periodic row of the transition metal (Cnd = 0 for 3d or 4d metal, 2 for 5d metal) as well as a correction for d(6) octahedral acids (Cd6 = 6 for d(6) metal ion in the acid, 0 for others) that are not dihydrogen complexes. Constants AL are provided for 13 commonly occurring ligand types; of these, nine neutral ligands are correlated with Lever's electrochemical ligand parameters EL. This method gives good estimates of the over 170 literature pKa values that range from less than zero to 50 with a standard deviation of 3 pKa units for complexes of the metals chromium to nickel, molybdenum, ruthenium to palladium, and tungsten to platinum in the periodic table. This approach allows a quick assessment of the acidity of hydride complexes found in nature (e.g., hydrogenases) and in industry (e.g., catalysis and hydrogen energy applications). The pKa values calculated for acids that have bulky or large bite angle chelating ligands deviate the most from this correlation. The method also provides an estimate of the base strength of the deprotonated form of the complex. PMID:24410025

  11. Detoxification of acidic biorefinery waste liquor for production of high value amino acid.

    PubMed

    Christopher, Meera; Anusree, Murali; Mathew, Anil K; Nampoothiri, K Madhavan; Sukumaran, Rajeev Kumar; Pandey, Ashok

    2016-08-01

    The current study evaluates the detoxification of acid pretreatment liquor (APL) using adsorbent (ADS 400 & ADS 800) or ion-exchange (A-27MP & A-72MP) resins and its potential for amino acid production. The APL is generated as a by-product from the pretreatment of lignocellulosic biomass and is rich monomeric sugars as well as sugar degradation products (fermentation inhibitors) such as furfural and hydroxymethyl furfural (HMF). Of the four resins compared, ADS 800 removed approximately 85% and 60% of furfural and HMF, respectively. ADS 800 could be reused for up to six cycles after regeneration without losing its adsorption properties. The study was further extended by assessing the fermentability of detoxified APL for l-lysine production using wild and mutant strains of Corynebacterium glutamicum. The detoxified APL was superior to APL for l-lysine production.

  12. Total alkalinity versus buffer value (capacity) as a sensitivity indicator for fresh waters receiving acid rain

    SciTech Connect

    Faust, S.D.

    1983-09-01

    The frequently stated idea that total alkalinity is a measure of the buffer capacity of a natural water is refuted. Total alkalinity is a measure of the acid neutralizing capacity, equivalents/liter, of a water. In natural waters, the carbonate system provides most of this neutralizing capacity. In as much as the pH values of natural fresh waters lie below 8.3, the total alkalinity is, for all intents and purposes, the total bicarbonate content. Any contributions of carbonate and hydroxide to total alkalinity are nil. The buffer capacity or buffer value is the relation between the increment of a strong base, or strong acid, that causes a one unit change in the pH value. The values of total alkalinity and pH, considered individually cannot give an accurate assessment of the impact of acid deposition on a natural water. Rather it is necessary to combine the pH and alkalinity values into the beta concept in order to assess accurately and to calculate the capacity of a natural water to resist the impact of acid deposition. An analytical determination of total alkalinity is given with an application of the beta value. 17 references, 2 figures, 4 tables.

  13. Study of the Affinity between the Protein Kinase PKA and Peptide Substrates Derived from Kemptide Using Molecular Dynamics Simulations and MM/GBSA

    PubMed Central

    Mena-Ulecia, Karel; Vergara-Jaque, Ariela; Poblete, Horacio; Tiznado, William; Caballero, Julio

    2014-01-01

    We have carried out a protocol in computational biochemistry including molecular dynamics (MD) simulations and MM/GBSA free energy calculations on the complex between the protein kinase A (PKA) and the specific peptide substrate Kemptide (LRRASLG). We made the same calculations on other PKA complexes that contain Kemptide derivatives (with mutations of the arginines, and with deletions of N and C-terminal amino acids). We predicted shifts in the free energy changes from the free PKA to PKA-substrate complex (ΔΔGE→ES) when Kemptide structure is modified (we consider that the calculated shifts correlate with the experimental shifts of the free energy changes from the free PKA to the transition states (ΔΔGE→TS) determined by the catalytic efficiency (kcat/KM) changes). Our results demonstrate that it is possible to predict the kinetic properties of protein kinases using simple computational biochemistry methods. As an additional benefit, these methods give detailed molecular information that permit the analysis of the atomic forces that contribute to the affinity between protein kinases and their substrates. PMID:25275314

  14. Study of the affinity between the protein kinase PKA and peptide substrates derived from kemptide using molecular dynamics simulations and MM/GBSA.

    PubMed

    Mena-Ulecia, Karel; Vergara-Jaque, Ariela; Poblete, Horacio; Tiznado, William; Caballero, Julio

    2014-01-01

    We have carried out a protocol in computational biochemistry including molecular dynamics (MD) simulations and MM/GBSA free energy calculations on the complex between the protein kinase A (PKA) and the specific peptide substrate Kemptide (LRRASLG). We made the same calculations on other PKA complexes that contain Kemptide derivatives (with mutations of the arginines, and with deletions of N and C-terminal amino acids). We predicted shifts in the free energy changes from the free PKA to PKA-substrate complex (ΔΔG(E→ES)) when Kemptide structure is modified (we consider that the calculated shifts correlate with the experimental shifts of the free energy changes from the free PKA to the transition states (ΔΔG(E→TS)) determined by the catalytic efficiency (k(cat)/K(M)) changes). Our results demonstrate that it is possible to predict the kinetic properties of protein kinases using simple computational biochemistry methods. As an additional benefit, these methods give detailed molecular information that permit the analysis of the atomic forces that contribute to the affinity between protein kinases and their substrates. PMID:25275314

  15. Computational study of the acid dissociation of esters and lactones. A case study of diketene.

    PubMed

    Gómez-Bombarelli, Rafael; González-Pérez, Marina; Pérez-Prior, María Teresa; Calle, Emilio; Casado, Julio

    2009-07-17

    A computational study of the aqueous pK(a) of some saturated and unsaturated cyclic and linear esters and ketones was carried out at the DFT-B3LYP 6-31++G(2df,2pd), CBS-Q, and G2 levels, with the integral equation formalism polarizable continuum model for solvation, using a proton exchange mechanism. The influence of unsaturation, position of the double bond, and cyclization were studied. The computational results show that (a) in all cases studied except that of diketene (4-methylene-2-oxetanone), the alpha-beta unsaturated isomer is 20-30 kJ mol(-1) lower in energy that the beta-gamma unsaturated one; (b) alpha-beta unsaturation lowers the pK(a) of an ester approximately 6 units, whereas beta-gamma unsaturation lowers it by approximately 10 units, and cyclization lowers the pK(a) by approximately 3 units. In order to check the predictive power of the methodology, the acid dissociation constant of diketene in water was measured via kinetic study of its base-catalyzed hydrolysis. The pK(a) value obtained (15.2 +/- 0.3) is in keeping with the expected value for a beta-gamma unsaturated beta-lactone. This low value also suggests that deprotonated diketene does not interconvert to a more stable, less acidic alpha-beta unsaturated isomer, which is also consistent with computational results. PMID:19459649

  16. Top value platform chemicals: bio-based production of organic acids.

    PubMed

    Becker, Judith; Lange, Anna; Fabarius, Jonathan; Wittmann, Christoph

    2015-12-01

    Driven by the quest for sustainability, recent years have seen a tremendous progress in bio-based production routes from renewable raw materials to commercial goods. Particularly, the production of organic acids has crystallized as a competitive and fast-evolving field, related to the broad applicability of organic acids for direct use, as polymer building blocks, and as commodity chemicals. Here, we review recent advances in metabolic engineering and industrial market scenarios with focus on organic acids as top value products from biomass, accessible through fermentation and biotransformation. PMID:26360870

  17. Top value platform chemicals: bio-based production of organic acids.

    PubMed

    Becker, Judith; Lange, Anna; Fabarius, Jonathan; Wittmann, Christoph

    2015-12-01

    Driven by the quest for sustainability, recent years have seen a tremendous progress in bio-based production routes from renewable raw materials to commercial goods. Particularly, the production of organic acids has crystallized as a competitive and fast-evolving field, related to the broad applicability of organic acids for direct use, as polymer building blocks, and as commodity chemicals. Here, we review recent advances in metabolic engineering and industrial market scenarios with focus on organic acids as top value products from biomass, accessible through fermentation and biotransformation.

  18. How Acidic Is Carbonic Acid?

    PubMed

    Pines, Dina; Ditkovich, Julia; Mukra, Tzach; Miller, Yifat; Kiefer, Philip M; Daschakraborty, Snehasis; Hynes, James T; Pines, Ehud

    2016-03-10

    Carbonic, lactic, and pyruvic acids have been generated in aqueous solution by the transient protonation of their corresponding conjugate bases by a tailor-made photoacid, the 6-hydroxy-1-sulfonate pyrene sodium salt molecule. A particular goal is to establish the pK(a) of carbonic acid H2CO3. The on-contact proton transfer (PT) reaction rate from the optically excited photoacid to the carboxylic bases was derived, with unprecedented precision, from time-correlated single-photon-counting measurements of the fluorescence lifetime of the photoacid in the presence of the proton acceptors. The time-dependent diffusion-assisted PT rate was analyzed using the Szabo-Collins-Kimball equation with a radiation boundary condition. The on-contact PT rates were found to follow the acidity order of the carboxylic acids: the stronger was the acid, the slower was the PT reaction to its conjugate base. The pK(a) of carbonic acid was found to be 3.49 ± 0.05 using both the Marcus and Kiefer-Hynes free energy correlations. This establishes H2CO3 as being 0.37 pK(a) units stronger and about 1 pK(a) unit weaker, respectively, than the physiologically important lactic and pyruvic acids. The considerable acid strength of intact carbonic acid indicates that it is an important protonation agent under physiological conditions. PMID:26862781

  19. Effect of surfactants on the dissociation constants of ascorbic and maleic acids.

    PubMed

    Jaiswal, P V; Ijeri, V S; Srivastava, A K

    2005-11-25

    The dissociation equilibria of ascorbic and maleic acids have been studied in certain cationic, anionic and non-ionic micellar media and their pK(a) values have been evaluated by the potentiometric, conductometric and spectrophotometric techniques. These pK(a) values have been found to shift in micellar media as compared to those in pure water. The differences in the values have been attributed to the solvent properties of the interfacial and bulk phases involving contribution from the micellar surface potential in the case of charged micelles. The values of the limiting molar conductance of the acids have been determined in the various surfactants and were found to be different in different surfactants. In case of the cationic surfactants the limiting molar conductance was found to increase with increase in surfactant concentration.

  20. Critical evaluation of buffering solutions for pKa determination by capillary electrophoresis.

    PubMed

    Fuguet, Elisabet; Reta, Mario; Gibert, Carme; Rosés, Martí; Bosch, Elisabeth; Ràfols, Clara

    2008-07-01

    The performance of the most common and also some other less common CE buffers has been tested for the pKa determination of several types of compounds (pyridine, amines, and phenols). The selected buffers cover a pH ranging from 3.7 to 11.8. Whereas some buffers, like acetic acid/acetate, BisTrisH+/BisTris, TrisH+/Tris, CHES/CHES-, and CAPS/CAPS- can be used with all type of analytes, others like ammonium/ammonia, butylammonium/butylammonia, ethylammonium/ethylammonia, diethylammonium/diethylammonia, and hydrogenphosphate/phosphate are not recommended because they interact with a wide range of compounds. The rest of the tested buffers (dihydrogenphosphate/hydrogenphosphate, MES/MES-, HEPES/HEPES-, and boric acid/borate) can show specific interactions depending on the nature of the analytes, and their use in some applications should be restricted.

  1. Impact of kinase activating and inactivating patient mutations on binary PKA interactions

    PubMed Central

    Röck, Ruth; Mayrhofer, Johanna E.; Bachmann, Verena; Stefan, Eduard

    2015-01-01

    The second messenger molecule cAMP links extracellular signals to intracellular responses. The main cellular cAMP effector is the compartmentalized protein kinase A (PKA). Upon receptor initiated cAMP-mobilization, PKA regulatory subunits (R) bind cAMP thereby triggering dissociation and activation of bound PKA catalytic subunits (PKAc). Mutations in PKAc or RIa subunits manipulate PKA dynamics and activities which contribute to specific disease patterns. Mutations activating cAMP/PKA signaling contribute to carcinogenesis or hormone excess, while inactivating mutations cause hormone deficiency or resistance. Here we extended the application spectrum of a Protein-fragment Complementation Assay based on the Renilla Luciferase to determine binary protein:protein interactions (PPIs) of the PKA network. We compared time- and dose-dependent influences of cAMP-elevation on mutually exclusive PPIs of PKAc with the phosphotransferase inhibiting RIIb and RIa subunits and the protein kinase inhibitor peptide (PKI). We analyzed PKA dynamics following integration of patient mutations into PKAc and RIa. We observed that oncogenic modifications of PKAc(L206R) and RIa(Δ184-236) as well as rare disease mutations in RIa(R368X) affect complex formation of PKA and its responsiveness to cAMP elevation. With the cell-based PKA PPI reporter platform we precisely quantified the mechanistic details how inhibitory PKA interactions and defined patient mutations contribute to PKA functions. PMID:26347651

  2. Development of Monopole Interaction Models for Ionic Compounds. Part I: Estimation of Aqueous Henry’s Law Constants for Ions and Gas Phase pKa Values for Acidic Compounds

    EPA Science Inventory

    The SPARC (SPARC Performs Automated Reasoning in Chemistry) physicochemical mechanistic models for neutral compounds have been extended to estimate Henry’s Law Constant (HLC) for charged species by incorporating ionic electrostatic interaction models. Combinations of absolute aq...

  3. Tonic PKA Activity Regulates SK Channel Nanoclustering and Somatodendritic Distribution.

    PubMed

    Abiraman, Krithika; Sah, Megha; Walikonis, Randall S; Lykotrafitis, George; Tzingounis, Anastasios V

    2016-06-01

    Small-conductance calcium-activated potassium (SK) channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK channels labeled with fluorophore-tagged apamin and monitored SK channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK channels were expressed in nanodomains as single entities or as a group of multiple channels. Our study reveals a new level of regulation of SK channels by cAMP-PKA and suggests that ion channel topography and nanoclustering might be under the control of second messenger cascades. PMID:27107637

  4. Tautomeric and Microscopic Protonation Equilibria of Anthranilic Acid and Its Derivatives.

    PubMed

    Zapała, Lidia; Woźnicka, Elżbieta; Kalembkiewicz, Jan

    2014-01-01

    The acid-base chemistry of three zwitterionic compounds, namely anthranilic (2-aminobenzoic acid), N-methylanthranilic and N-phenylanthranilic acid has been characterized in terms of the macroconstants Ka1, Ka2, the isoelectric point pHI, the tautomerization constant Kz and microconstants k11, k12, k21, k22. The potentiometric titration method was used to determine the macrodissociation constants. Due to the very poor water solubility of N-phenylanthranilic acid the dissociation constants pKa1 and pKa2 were determined in MDM-water mixtures [MDM is a co-solvent mixture, consisting of equal volumes of methanol (MeOH), dioxane and acetonitrile (MeCN)]. The Yasuda-Shedlovsky extrapolation procedure has been used to obtain the values of pKa1 and pKa2 in aqueous solutions. The pKa1 and pKa2 values obtained by this method are 2.86 ± 0.01 and 4.69 ± 0.03, respectively. The tautomerization constant Kz describing the equilibrium between unionized form ⇌ zwitterionic form was evaluated by the Kz method based on UV-VIS spectrometry. The method uses spectral differences between the zwitterionic form (found at isoelectric pH in aqueous solution) and the unionized form (formed in an organic solvent of low dielectric constant). The highest value of the Kz constant has been observed in the case of N-methylantranilic acid (log10Kz = 1.31 ± 0.04). The values of log10Kz for anthranilic and N-phenylanthranilic acids are similar and have values of 0.93 ± 0.03 and 0.90 ± 0.05, respectively. The results indicate that the tested compounds, in aqueous solution around the isoelectric point pHI, occur mainly in the zwitterionic form. Moreover, the influence of the type of substituent and pH of the aqueous phase on the equilibrium were analyzed with regard to the formation and the coexistence of different forms of the acids in the examined systems.

  5. Determination of free fatty acids in pharmaceutical lipids by ¹H NMR and comparison with the classical acid value.

    PubMed

    Skiera, Christina; Steliopoulos, Panagiotis; Kuballa, Thomas; Diehl, Bernd; Holzgrabe, Ulrike

    2014-05-01

    Indices like acid value, peroxide value, and saponification value play an important role in quality control and identification of lipids. Requirements on these parameters are given by the monographs of the European pharmacopeia. (1)H NMR spectroscopy provides a fast and simple alternative to these classical approaches. In the present work a new (1)H NMR approach to determine the acid value is described. The method was validated using a statistical approach based on a variance components model. The performance under repeatability and in-house reproducibility conditions was assessed. We applied this (1)H NMR assay to a wide range of different fatty oils. A total of 305 oil and fat samples were examined by both the classical and the NMR method. Except for hard fat, the data obtained by the two methods were in good agreement. The (1)H NMR method was adapted to analyse waxes and oleyloleat. Furthermore, the effect of solvent and in the case of castor oil the effect of the oil matrix on line broadening and chemical shift of the carboxyl group signal are discussed.

  6. Determination of free fatty acids in pharmaceutical lipids by ¹H NMR and comparison with the classical acid value.

    PubMed

    Skiera, Christina; Steliopoulos, Panagiotis; Kuballa, Thomas; Diehl, Bernd; Holzgrabe, Ulrike

    2014-05-01

    Indices like acid value, peroxide value, and saponification value play an important role in quality control and identification of lipids. Requirements on these parameters are given by the monographs of the European pharmacopeia. (1)H NMR spectroscopy provides a fast and simple alternative to these classical approaches. In the present work a new (1)H NMR approach to determine the acid value is described. The method was validated using a statistical approach based on a variance components model. The performance under repeatability and in-house reproducibility conditions was assessed. We applied this (1)H NMR assay to a wide range of different fatty oils. A total of 305 oil and fat samples were examined by both the classical and the NMR method. Except for hard fat, the data obtained by the two methods were in good agreement. The (1)H NMR method was adapted to analyse waxes and oleyloleat. Furthermore, the effect of solvent and in the case of castor oil the effect of the oil matrix on line broadening and chemical shift of the carboxyl group signal are discussed. PMID:23664852

  7. Photometric method for determination of acidity constants through integral spectra analysis.

    PubMed

    Zevatskiy, Yuriy Eduardovich; Ruzanov, Daniil Olegovich; Samoylov, Denis Vladimirovich

    2015-04-15

    An express method for determination of acidity constants of organic acids, based on the analysis of the integral transmittance vs. pH dependence is developed. The integral value is registered as a photocurrent of photometric device simultaneously with potentiometric titration. The proposed method allows to obtain pKa using only simple and low-cost instrumentation. The optical part of the experimental setup has been optimized through the exclusion of the monochromator device. Thus it only takes 10-15 min to obtain one pKa value with the absolute error of less than 0.15 pH units. Application limitations and reliability of the method have been tested for a series of organic acids of various nature.

  8. Photometric method for determination of acidity constants through integral spectra analysis

    NASA Astrophysics Data System (ADS)

    Zevatskiy, Yuriy Eduardovich; Ruzanov, Daniil Olegovich; Samoylov, Denis Vladimirovich

    2015-04-01

    An express method for determination of acidity constants of organic acids, based on the analysis of the integral transmittance vs. pH dependence is developed. The integral value is registered as a photocurrent of photometric device simultaneously with potentiometric titration. The proposed method allows to obtain pKa using only simple and low-cost instrumentation. The optical part of the experimental setup has been optimized through the exclusion of the monochromator device. Thus it only takes 10-15 min to obtain one pKa value with the absolute error of less than 0.15 pH units. Application limitations and reliability of the method have been tested for a series of organic acids of various nature.

  9. Determination of acid-base dissociation constants of very weak zwitterionic heterocyclic bases by capillary zone electrophoresis.

    PubMed

    Ehala, Sille; Grishina, Anastasiya A; Sheshenev, Andrey E; Lyapkalo, Ilya M; Kašička, Václav

    2010-12-17

    Thermodynamic acid-base dissociation (ionization) constants (pK(a)) of seven zwitterionic heterocyclic bases, first representatives of new heterocyclic family (2,3,5,7,8,9-hexahydro-1H-diimidazo[1,2-c:2',1'-f][1,3,2]diazaphosphinin-4-ium-5-olate 5-oxides), originally designed as chiral Lewis base catalysts for enantioselective reactions, were determined by capillary zone electrophoresis (CZE). The pK(a) values of the above very weak zwitterionic bases were determined from the dependence of their effective electrophoretic mobility on pH in strongly acidic background electrolytes (pH 0.85-2.80). Prior to pK(a) calculation by non-linear regression analysis, the CZE measured effective mobilities were corrected to reference temperature, 25°C, and constant ionic strength, 25 mM. Thermodynamic pK(a) values of the analyzed zwitterionic heterocyclic bases were found to be particularly low, in the range 0.04-0.32. Moreover, from the pH dependence of effective mobility of the bases, some other relevant characteristics, such as actual and absolute ionic mobilities and hydrodynamic radii of the acidic cationic forms of the bases were determined.

  10. Influence of subcascade formation on displacement damage at high PKA energies

    SciTech Connect

    Stoller, R.E.; Greenwood, L.R.

    1997-08-01

    The design of first generation fusion reactors will have to be rely on radiation effects data obtained from experiments conducted in fission reactors. Two issues must be addressed to use this data with confidence. The first is differences in the neutron energy spectrum, and the second is differences in nuclear transmutation rates. Differences in the neutron energy spectra are reflected in the energy spectra of the primary knockon atoms (PKA). The issue of PKA energy effects has been addressed through the use of displacement cascade simulations using the method of molecular dynamics (MD). Although MD simulations can provide a detailed picture of the formation and evolution of displacement cascades, they impose a substantial computational burden. However, recent advances in computing equipment permit the simulation of high energy displacement events involving more than one-million atoms; the results presented here encompass MD cascade simulation energies from near the displacement threshold to as high as 40 keV. Two parameters have been extracted from the MD simulations: the number of point defects that remain after the displacement event is completed and the fraction of the surviving interstitials that are contained in clusters. The MD values have been normalized to the number of atomic displacements calculated with the secondary displacement model by Norgett, Robinson, and Torrens (NRT).

  11. Determination of the dissociation constants (pKa) of secondary and tertiary amines in organic media by capillary electrophoresis and their role in the electrophoretic mobility order inversion.

    PubMed

    Cantu, Marcelo Delmar; Hillebranda, Sandro; Carrilho, Emanuel

    2005-03-11

    Non-aqueous capillary electrophoresis (NACE) may provide a selectivity enhancement in separations since the analyte dissociation constants (pKa) in organic media are different from those in aqueous solutions. In this work, we have studied the inversion in mobility order observed in the separation of tertiary (imipramine (IMI) and amitryptiline (AMI)) and secondary amines (desipramine (DES) and nortryptiline (NOR)) in water, methanol, and acetonitrile. We have determined the pKa values in those solvents and the variation of dissociation constants with the temperature. From these data, and applying the Van't Hoff equation, we have calculated the thermodynamic parameters deltaH and deltaS. The pKa values found in methanol for DES, NOR, IMI, and AMI were 10.80, 10.79, 10.38, and 10.33, respectively. On the other hand, in acetonitrile an opposite relation was found since the values were 20.60, 20.67, 20.74, and 20.81 for DES, NOR, IMI, and AMI. This is the reason why a migration order inversion is observed in NACE for these solvents. The thermodynamic parameters were evaluated and presented a tendency that can be correlated with that observed for pKa values.

  12. Study on analysis of waste edible oil with deterioration and removal of acid value, carbonyl value, and free fatty acid by a food additive (calcium silicate).

    PubMed

    Ogata, Fumihiko; Tanaka, Yuko; Tominaga, Hisato; Kangawa, Moe; Inoue, Kenji; Ueda, Ayaka; Iwata, Yuka; Kawasaki, Naohito

    2013-01-01

    This study investigated the regeneration of waste edible oil using a food additive (calcium silicate, CAS). Waste edible oil was prepared by combined heat and aeration treatment. Moreover, the deterioration of edible oil by combined heat and aeration treatment was greater than that by heat treatment alone. The acid value (AV) and carbonyl value (CV) increased with increasing deterioration; conversely, the tocopherol concentration decreased with increasing deterioration. The specific surface area, pore volume, and mean pore diameter of the 3 CAS formulations used (CAS30, CAS60, and CAS90) were evaluated, and scanning electron microscopic images were taken. The specific surface area increased in the order of CAS30 (115.54 m(2)/g) < CAS60 (163.93 m(2)/g) < CAS90 (187.47 m(2)/g). The mean pore diameter increased in the order of CAS90 (170.59 Å) < CAS60 (211.60 Å) < CAS30 (249.70 Å). The regeneration of waste edible oil was possible with CAS treatment. The AV reduced by 15.2%, 10.8%, and 23.1% by CAS30, CAS60, and CAS90 treatment, respectively, and the CV was reduced by 35.6%, 29.8%, and 31.3% by these 3 treatments, respectively. Moreover, the concentrations of tocopherol and free fatty acids did not change with CAS treatment. The characteristics of CAS were not related to the degree of change of AV and CV. However, the adsorption mechanism of polar and non-polar compounds generated in waste edible oil by CAS was related with the presence of silica gel molecules in CAS. The findings indicated that CAS was useful for the regeneration of waste edible oil.

  13. Progresses in Ab Initio QM/MM Free Energy Simulations of Electrostatic Energies in Proteins: Accelerated QM/MM Studies of pKa, Redox Reactions and Solvation Free Energies

    SciTech Connect

    Kamerlin, Shina C. L.; Haranczyk, Maciej; Warshel, Arieh

    2009-03-01

    Hybrid quantum mechanical / molecular mechanical (QM/MM) approaches have been used to provide a general scheme for chemical reactions in proteins. However, such approaches still present a major challenge to computational chemists, not only because of the need for very large computer time in order to evaluate the QM energy but also because of the need for propercomputational sampling. This review focuses on the sampling issue in QM/MM evaluations of electrostatic energies in proteins. We chose this example since electrostatic energies play a major role in controlling the function of proteins and are key to the structure-function correlation of biological molecules. Thus, the correct treatment of electrostatics is essential for the accurate simulation of biological systems. Although we will be presenting here different types of QM/MM calculations of electrostatic energies (and related properties), our focus will be on pKa calculations. This reflects the fact that pKa of ionizable groups in proteins provide one of the most direct benchmarks for the accuracy of electrostatic models of macromolecules. While pKa calculations by semimacroscopic models have given reasonable results in many cases, existing attempts to perform pKa calculations using QM/MM-FEP have led to large discrepancies between calculated and experimental values. In this work, we accelerate our QM/MM calculations using an updated mean charge distribution and a classical reference potential. We examine both a surface residue (Asp3) of the bovine pancreatic trypsin inhibitor, as well as a residue buried in a hydrophobic pocket (Lys102) of the T4-lysozyme mutant. We demonstrate that by using this approach, we are able to reproduce the relevant sidechain pKas with an accuracy of 3 kcal/mol. This is well within the 7 kcal/mol energy difference observed in studies of enzymatic catalysis, and is thus sufficient accuracy to determine the main contributions to the catalytic energies of enzymes. We also provide an

  14. pH-Metric determination of acid values in oilseeds without titration.

    PubMed

    Kuselman, I; Tur'yan, Y I; Burenko, T; Goldfeld, I; Anisimov, B

    1999-07-01

    A new pH-metric method for determination of acid values in oilseeds without titration has been developed in the range 0.6-10 and more mg KOH/g. The method is based on a rapid (1-2 min) selective and complete extraction of free fatty acids from an oilseed test portion into a special reagent A, separation of the solution from the solid oilseed material by centrifugation or filtration, transfer of an aliquot of the solution into a pH-metric cell with reagent B for measurement of conditional pH(1)' of the formed mixture, addition of standard acid (HCl or H(2)SO(4)) and pH(2)' measurement. The reagents are non-toxic, and the method is rapid. Its metrological parameters for Soybean, Canola and Sunflower oilseeds are satisfactory for practical purposes.

  15. PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development.

    PubMed

    Drelon, Coralie; Berthon, Annabel; Sahut-Barnola, Isabelle; Mathieu, Mickaël; Dumontet, Typhanie; Rodriguez, Stéphanie; Batisse-Lignier, Marie; Tabbal, Houda; Tauveron, Igor; Lefrançois-Martinez, Anne-Marie; Pointud, Jean-Christophe; Gomez-Sanchez, Celso E; Vainio, Seppo; Shan, Jingdong; Sacco, Sonia; Schedl, Andreas; Stratakis, Constantine A; Martinez, Antoine; Val, Pierre

    2016-01-01

    Adrenal cortex physiology relies on functional zonation, essential for production of aldosterone by outer zona glomerulosa (ZG) and glucocorticoids by inner zona fasciculata (ZF). The cortex undergoes constant cell renewal, involving recruitment of subcapsular progenitors to ZG fate and subsequent lineage conversion to ZF identity. Here we show that WNT4 is an important driver of WNT pathway activation and subsequent ZG differentiation and demonstrate that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. Furthermore, we provide evidence that constitutive PKA activation inhibits, whereas partial inactivation of PKA catalytic activity stimulates β-catenin-induced tumorigenesis. Together, both lower PKA activity and higher WNT pathway activity lead to poorer prognosis in adrenocortical carcinoma (ACC) patients. These observations suggest that PKA acts as a tumour suppressor in the adrenal cortex, through repression of WNT signalling. PMID:27624192

  16. PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development

    PubMed Central

    Drelon, Coralie; Berthon, Annabel; Sahut-Barnola, Isabelle; Mathieu, Mickaël; Dumontet, Typhanie; Rodriguez, Stéphanie; Batisse-Lignier, Marie; Tabbal, Houda; Tauveron, Igor; Lefrançois-Martinez, Anne-Marie; Pointud, Jean-Christophe; Gomez-Sanchez, Celso E.; Vainio, Seppo; Shan, Jingdong; Sacco, Sonia; Schedl, Andreas; Stratakis, Constantine A.; Martinez, Antoine; Val, Pierre

    2016-01-01

    Adrenal cortex physiology relies on functional zonation, essential for production of aldosterone by outer zona glomerulosa (ZG) and glucocorticoids by inner zona fasciculata (ZF). The cortex undergoes constant cell renewal, involving recruitment of subcapsular progenitors to ZG fate and subsequent lineage conversion to ZF identity. Here we show that WNT4 is an important driver of WNT pathway activation and subsequent ZG differentiation and demonstrate that PKA activation prevents ZG differentiation through WNT4 repression and WNT pathway inhibition. This suggests that PKA activation in ZF is a key driver of WNT inhibition and lineage conversion. Furthermore, we provide evidence that constitutive PKA activation inhibits, whereas partial inactivation of PKA catalytic activity stimulates β-catenin-induced tumorigenesis. Together, both lower PKA activity and higher WNT pathway activity lead to poorer prognosis in adrenocortical carcinoma (ACC) patients. These observations suggest that PKA acts as a tumour suppressor in the adrenal cortex, through repression of WNT signalling. PMID:27624192

  17. A Targeted Mutation Identified through pKa Measurements Indicates a Postrecruitment Role for Fis1 in Yeast Mitochondrial Fission.

    PubMed

    Koppenol-Raab, Marijke; Harwig, Megan Cleland; Posey, Ammon E; Egner, John M; MacKenzie, Kevin R; Hill, R Blake

    2016-09-23

    The tail-anchored protein Fis1 is implicated as a passive tether in yeast mitochondrial fission. We probed the functional role of Fis1 Glu-78, whose elevated side chain pKa suggests participation in protein interactions. Fis1 binds partners Mdv1 or Dnm1 tightly, but mutation E78A weakens Fis1 interaction with Mdv1, alters mitochondrial morphology, and abolishes fission in a growth assay. In fis1Δ rescue experiments, Fis1-E78A causes a novel localization pattern in which Dnm1 uniformly coats the mitochondria. By contrast, Fis1-E78A at lower expression levels recruits Dnm1 into mitochondrial punctate structures but fails to support normal fission. Thus, Fis1 makes multiple interactions that support Dnm1 puncta formation and may be essential after this step, supporting a revised model for assembly of the mitochondrial fission machinery. The insights gained by mutating a residue with a perturbed pKa suggest that side chain pKa values inferred from routine NMR sample pH optimization could provide useful leads for functional investigations.

  18. The catalytic role of aspartic acid-92 in a human dual-specific protein-tyrosine-phosphatase.

    PubMed

    Denu, J M; Zhou, G; Guo, Y; Dixon, J E

    1995-03-14

    The mechanism of catalysis for the human dual-specific (vaccinia H1-related) protein-tyrosine-phosphatase was investigated. The pH dependence of the kcat value is bell-shaped when p-nitrophenyl phosphate was employed as a model substrate. The kcat/Km pH profile rises with a slope of 2 and decreases with a slope of -1, indicating that two groups must be unprotonated and one group must be protonated for activity. An amino acid residue with an apparent pKa value of 5.5 +/- 0.2 must be unprotonated and a residue with a pKa value of 5.7 must be unprotonated for activity. The pKa value of the catalytic cysteine-124 (C124) was 5.6 +/- 0.1. The aspartic acid-92-asparagine (D92N) mutant enzyme was 100-fold less active than the native enzyme and exhibited the loss of the basic limb in the pH profiles, suggesting that in the native enzyme D92 must be protonated for activity. The D92 residue is conserved throughout the entire family of dual-specific phosphatases. Mutants glutamic acid-6-glutamine, glutamic acid-32-glutamine, aspartic acid-14-asparagine, and aspartic acid-110-asparagine had less than a 2-fold effect on the kinetic parameters when compared to native enzyme. Based upon the lack of a "burst" in rapid reaction kinetics, formation of the intermediate is rate-limiting with both native and D92N mutant enzymes. In agreement with rate-limiting formation of the intermediate, the pKa value of 5.5 for the group which must be unprotonated for activity was assigned to C124.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Characterization and Diagnostic Value of Amino Acid Side Chain Neutral Losses Following Electron-Transfer Dissociation

    PubMed Central

    Xia, Qiangwei; Lee, M. Violet; Rose, Christopher M.; Marsh, Alyce J.; Hubler, Shane L.; Wenger, Craig D.; Coon, Joshua J.

    2011-01-01

    Using a large set of high mass accuracy and resolution ETD tandem mass spectra, we characterized ETD-induced neutral losses. From these data we deduced the chemical formula for 20 of these losses. Many of them have been previously observed in electron-capture dissociation (ECD) spectra, such as losses of the side chains of arginine, aspartic acid, glutamic acid, glutamine, asparagine, leucine, histidine, and carbamidomethylated cysteine residues. With this information, we examined the diagnostic value of these amino acid-specific losses. Among 1285 peptide–spectrum matches, 92.5% have agreement between neutral loss-derived peptide amino acid composition and the peptide sequences. Moreover, we show that peptides can be uniquely identified by using only the accurate precursor mass and amino acid composition based on neutral losses; the median number of sequence candidates from an accurate mass query is reduced from 21 to 8 by adding side chain loss information. Besides increasing confidence in peptide identification, our findings suggest the potential use of these diagnostic losses in ETD spectra to improve false discovery rate estimation and to enhance the performance of scoring functions in database search algorithms. PMID:21472585

  20. Characterization and Diagnostic Value of Amino Acid Side Chain Neutral Losses Following Electron-Transfer Dissociation

    NASA Astrophysics Data System (ADS)

    Xia, Qiangwei; Lee, M. Violet; Rose, Christopher M.; Marsh, Alyce J.; Hubler, Shane L.; Wenger, Craig D.; Coon, Joshua J.

    2011-02-01

    Using a large set of high mass accuracy and resolution ETD tandem mass spectra, we characterized ETD-induced neutral losses. From these data we deduced the chemical formula for 20 of these losses. Many of them have been previously observed in electron-capture dissociation (ECD) spectra, such as losses of the side chains of arginine, aspartic acid, glutamic acid, glutamine, asparagine, leucine, histidine, and carbamidomethylated cysteine residues. With this information, we examined the diagnostic value of these amino acid-specific losses. Among 1285 peptide-spectrum matches, 92.5% have agreement between neutral loss-derived peptide amino acid composition and the peptide sequences. Moreover, we show that peptides can be uniquely identified by using only the accurate precursor mass and amino acid composition based on neutral losses; the median number of sequence candidates from an accurate mass query is reduced from 21 to 8 by adding side chain loss information. Besides increasing confidence in peptide identification, our findings suggest the potential use of these diagnostic losses in ETD spectra to improve false discovery rate estimation and to enhance the performance of scoring functions in database search algorithms.

  1. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor: EFFECT ON PKA LOCALIZATION AND P-Rex1 SIGNALING.

    PubMed

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G H; Fukuhara, Shigetomo; Taylor, Susan S; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-03-18

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. PMID:26797121

  2. Promotion of PDGF-induced endothelial cell migration by phosphorylated VASP depends on PKA anchoring via AKAP.

    PubMed

    Zhang, Deling; Ouyang, Jingping; Wang, Nian; Zhang, Yahui; Bie, Jinghua; Zhang, Yemin

    2010-02-01

    Vasodilator-stimulated phosphoprotein (VASP), an important substrate of PKA, plays a critical role in remodeling of actin cytoskeleton and actin-based cell motility. However, how PKA accurately transfers extracellular signals to VASP and then how phosphorylation of VASP regulates endothelial cell migration have not been clearly defined. Protein kinase A anchoring proteins (AKAPs) are considered to regulate intracellular-specific signal targeting of PKA via AKAP-mediated PKA anchoring. Thus, our study investigated the relationship among AKAP anchoring of PKA, PKA activity, and VASP phosphorylation, which is to clarify the exact role of VASP and its upstream regulatory mechanism in PKA-dependent migration. Our results show that chemotactic factor PDGF activated PKA, increased phosphorylation of VASP at Ser157, and enhanced ECV304 endothelial cell migration. However, phosphorylation site-directed mutation of VASP at Ser157 attenuated the chemotactic effect of PDGF on endothelial cells, suggesting phosphorylation of VASP at Ser157 promotes PKA-mediated endothelial cell migration. Furthermore, disrupting PKA anchoring to AKAP or PKA activity significantly attenuated the PKA activity, VASP phosphorylation, and subsequent cell migration. Meanwhile, disrupting PKA anchoring to AKAP abolished PDGF-induced lamellipodia formation and special VASP accumulation at leading edge of lamellipodia. These results indicate that PKA activation and PKA-mediated substrate responses in VASP phosphorylation and localization depend on PKA anchoring via AKAP in PDGF-induced endothelial cell migration. In conclusion, AKAP anchoring of PKA is an essential upstream event in regulation of PKA-mediated VASP phosphorylation and subsequent endothelial cell migration, which contributes to explore new methods for controlling endothelial cell migration related diseases and angiogenesis.

  3. [Effect of extrusion on the retention of amino acids and the nutritional value of the protein].

    PubMed

    Martinchik, A N; Sharikov, A Yu

    2015-01-01

    The data of the literature on the impact factors of the extrusion cooking on physical and chemical properties of food proteins, biological value and digestibility have been discussed. Extrusion cooking is a high temperature short-time process, characterizing by a minimal loss of nutrients. and biologically active substances compared to other methods of heat treatment of food. Studies of the properties of protein-containing products, protein isolates and concentrates in the extrusion are examined in different ways: the inactivation of antinutritional factors; improvement in digestibility and availability; changes in the content and chemical modification of amino acids; Maillard reactions involving amino acids; mutual enrichment of protein mixtures during the composite food extrusion; formation of functional technological properties of the extruded protein products.

  4. Unified pH values of liquid chromatography mobile phases.

    PubMed

    Suu, Agnes; Jalukse, Lauri; Liigand, Jaanus; Kruve, Anneli; Himmel, Daniel; Krossing, Ingo; Rosés, Martí; Leito, Ivo

    2015-03-01

    This work introduces a conceptually new approach of measuring pH of mixed-solvent liquid chromatography (LC) mobile phases. Mobile phase pH is very important in LC, but its correct measurement is not straightforward, and all commonly used approaches have deficiencies. The new approach is based on the recently introduced unified pH (pH(abs)) scale, which enables direct comparison of acidities of solutions made in different solvents based on chemical potential of the proton in the solutions. This work represents the first experimental realization of the pH(abs) concept using differential potentiometric measurement for comparison of the chemical potentials of the proton in different solutions (connected by a salt bridge), together with earlier published reference points for obtaining the pH(abs) values (referenced to the gas phase) or pH(abs)(H₂O) values (referenced to the aqueous solution). The liquid junction potentials were estimated in the framework of Izutsu's three-component method. pH(abs) values for a number of common LC and LC-MS mobile phases have been determined. The pH(abs) scale enables for the first time direct comparison of acidities of any LC mobile phases, with different organic additives, different buffer components, etc. A possible experimental protocol of putting this new approach into chromatographic practice has been envisaged and its applicability tested. It has been demonstrated that the ionization behavior of bases (cationic acids) in the mobile phases can be better predicted by using the pH(abs)(H₂O) values and aqueous pKa values than by using the alternative means of expressing mobile phase acidity. Description of the ionization behavior of acids on the basis of pH(abs)(H₂O) values is possible if the change of their pKa values with solvent composition change is taken into account. PMID:25664372

  5. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA.

    PubMed

    Angulo-Rojo, Carla; Manning-Cela, Rebeca; Aguirre, Adán; Ortega, Arturo; López-Bayghen, Esther

    2013-12-23

    The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJ(κ) pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision.

  6. Involvement of the Notch pathway in terminal astrocytic differentiation: role of PKA

    PubMed Central

    Angulo-Rojo, Carla; Manning-Cela, Rebeca; Aguirre, Adán; Ortega, Arturo; López-Bayghen, Esther

    2013-01-01

    The Notch pathway is a highly conserved signaling system essential for modulating neurogenesis and promoting astrogenesis. Similarly, the cAMP signaling cascade can promote astrocytic commitment in several cell culture models, such as the C6 glioma cell line. These cells have the capacity to differentiate into oligodendrocytes or astrocytes, characteristics that allow their use as a glial progenitor model. In this context, we explore here the plausible involvement of cAMP in Notch-dependent signal transactions. The exposure of C6 cells to a non-hydrolysable cAMP analogue resulted in a sustained augmentation of Notch activity, as detected by nuclear translocation of its intracellular domain portion (NICD) and transcriptional activity. The cAMP effect is mediated through the activation of the γ-secretase complex, responsible for Notch cleavage and is sensitive to inhibitors of the cAMP-dependent protein kinase, PKA. As expected, Notch cleavage and nuclear translocation resulted in the up-regulation of the mRNA levels of one of its target genes, the transcription factor Hair and enhancer of split 5. Moreover, the glutamate uptake activity, as well as the expression of astrocytic markers such as glial fibrillary acidic protein, S100β protein and GLAST was also enhanced in cAMP-exposed cells. Our results clearly suggest that during the process of C6 astrocytic differentiation, cAMP activates the PKA/γ-secretase/NICD/RBPJκ pathway and Notch1 expression, leading to transcriptional activation of the genes responsible for glial progenitor cell fate decision. PMID:24286475

  7. A computational study of ultrafast acid dissociation and acid-base neutralization reactions. I. The model.

    PubMed

    Maurer, Patrick; Thomas, Vibin; Rivard, Ugo; Iftimie, Radu

    2010-07-28

    Ultrafast, time-resolved investigations of acid-base neutralization reactions have recently been performed using systems containing the photoacid 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) and various Bronsted bases. Two conflicting neutralization mechanisms have been formulated by Mohammed et al. [Science 310, 83 (2005)] and Siwick et al. [J. Am. Chem. Soc. 129, 13412 (2007)] for the same acid-base system. Herein an ab initio molecular dynamics based computational model is formulated, which is able to investigate the validity of the proposed mechanisms in the general context of ground-state acid-base neutralization reactions. Our approach consists of using 2,4,6-tricyanophenol (exp. pKa congruent with 1) as a model for excited-state HPTS( *) (pKa congruent with 1.4) and carboxylate ions for the accepting base. We employ our recently proposed dipole-field/quantum mechanics (QM) treatment [P. Maurer and R. Iftimie, J. Chem. Phys. 132, 074112 (2010)] of the proton donor and acceptor molecules. This approach allows one to tune the free energy of neutralization to any desired value as well as model initial nonequilibrium hydration effects caused by a sudden increase in acidity, making it possible to achieve a more realistic comparison with experimental data than could be obtained via a full-QM treatment of the entire system. It is demonstrated that the dipole-field/QM model reproduces correctly key properties of the 2,4,6-tricyanophenol acid molecule including gas-phase proton dissociation energies and dipole moments, and condensed-phase hydration structure and pKa values.

  8. Estimation of the acid dissociation constant of perfluoroalkyl carboxylic acids through an experimental investigation of their water-to-air transport.

    PubMed

    Vierke, Lena; Berger, Urs; Cousins, Ian T

    2013-10-01

    The acid dissociation constants (pKas) of perfluoroalkyl carboxylic acids (PFCAs) have been the subject of discussion in the literature; for example, values from -0.2 to 3.8 have been suggested for perfluorooctanoic acid (PFOA). The dissociated anionic conjugate bases of PFCAs have negligible air-water partition coefficients (KAWs) and do not volatilize from water. The neutral acids, however, have relatively high KAWs and volatilization from water has been demonstrated. The extent of volatilization of PFCAs in the environment will depend on the water pH and their pKa. Knowledge of the pKas of PFCAs is therefore vital for understanding their environmental transport and fate. We investigated the water-to-air transfer of PFCAs in a novel experimental setup. We used ∼1 μg L(-1) of PFCAs in water (above environmental background concentrations but below the concentration at which self-association occurs) at different water pH (pH 0.3 to pH 6.9) and sampled the PFCAs volatilized from water during a 2-day experiment. Our results suggest that the pKas of C4-11 PFCAs are <1.6. For PFOA, we derived a pKa of 0.5 from fitting the experimental measurements with a volatilization model. Perfluoroalkane sulfonic acids were not volatilized, suggesting that their pKas are below the investigated pH range (pKa <0.3).

  9. Temperature dependence of interfacial structures and acidity of clay edge surfaces

    NASA Astrophysics Data System (ADS)

    Liu, Xiandong; Lu, Xiancai; Cheng, Jun; Sprik, Michiel; Wang, Rucheng

    2015-07-01

    In the pursuit of a microscopic understanding of the effects of temperature on the surface reactivity of clay minerals, we conducted first principles molecular dynamics (FPMD) simulations to study the interfacial structures and acidity of clay edge surfaces at elevated temperatures. The common edge surfaces ((0 1 0) and (1 1 0) types) of phyllosilicates were investigated at 348 K and 423 K, and the results were compared with those previously derived at ambient conditions. We found that the stable surface sites are the same as at ambient conditions, including tbnd Al(OH2)2 (6-fold Al), tbnd Al(OH2) (5-fold Al) and tbnd Si(OH) on the (0 1 0) facet, and tbnd Al(OH2), tbnd Al(OH)Sitbnd and tbnd Si(OH) on the (1 1 0) surface. The FPMD-based vertical energy gap technique was applied to compute the acidity constants of edge sites and the resulting pKa values show a decreasing trend with temperature. The results demonstrate that although changes in the point of zero charge of the entire material are insignificant up to 348 K, the decrease in surface pKa can be 3 pKa units, while it can be as large as 6 pKa units up to 423 K. The derived interface structures and pKa values can be used in future experimental and modeling research, e.g., in interpreting experiments and predicting the surface complexation of metal cations and organics. This study therefore provides a physical basis for investigating the interfacial processes of clay minerals in environments that experience elevated P-T conditions, such as sedimentary basins and geological nuclear waste repositories.

  10. The value of short amino acid sequence matches for prediction of protein allergenicity.

    PubMed

    Silvanovich, Andre; Nemeth, Margaret A; Song, Ping; Herman, Rod; Tagliani, Laura; Bannon, Gary A

    2006-03-01

    Typically, genetically engineered crops contain traits encoded by one or a few newly expressed proteins. The allergenicity assessment of newly expressed proteins is an important component in the safety evaluation of genetically engineered plants. One aspect of this assessment involves sequence searches that compare the amino acid sequence of the protein to all known allergens. Analyses are performed to determine the potential for immunologically based cross-reactivity where IgE directed against a known allergen could bind to the protein and elicit a clinical reaction in sensitized individuals. Bioinformatic searches are designed to detect global sequence similarity and short contiguous amino acid sequence identity. It has been suggested that potential allergen cross-reactivity may be predicted by identifying matches as short as six to eight contiguous amino acids between the protein of interest and a known allergen. A series of analyses were performed, and match probabilities were calculated for different size peptides to determine if there was a scientifically justified search window size that identified allergen sequence characteristics. Four probability modeling methods were tested: (1) a mock protein and a mock allergen database, (2) a mock protein and genuine allergen database, (3) a genuine allergen and genuine protein database, and (4) a genuine allergen and genuine protein database combined with a correction for repeating peptides. These analyses indicated that searches for short amino acid sequence matches of eight amino acids or fewer to identify proteins as potential cross-reactive allergens is a product of chance and adds little value to allergy assessments for newly expressed proteins.

  11. NMR assignments and the acid-base characterization of the pomegranate ellagitannin punicalagin in the acidic pH-range.

    PubMed

    Kraszni, Márta; Marosi, Attila; Larive, Cynthia K

    2013-07-01

    In exploring the capability of nuclear magnetic resonance (NMR) spectroscopy for pomegranate juice analysis, the eight aromatic singlet resonances of α- and β-punicalagin were clearly identified in the (1)H NMR spectra of juice samples. The four downfield resonances were found to be sensitive to small pH changes around pH 3.50 where the NMR spectra of the juice samples were recorded. To understand this unusual behavior, the (1)H and (13)C resonance assignments of the punicalagin anomers were determined in aqueous solution and pH titrations with UV and (1)H NMR detection carried out to characterize the acid-base properties of punicalagin over the pH range 2-8. Simultaneous fitting of all of the pH-sensitive (1)H NMR signals produced similar but significantly different pKa values for the first two deprotonation equilibria of the gallagic acid moiety of the punicalagin α- (pKa1 = 4.57 ± 0.02, pKa2 = 5.63 ± 0.03) and β- (pKa1 = 4.36 ± 0.01, pKa2 = 5.47 ± 0.02) anomers. Equivalent pKa values, (α : 6.64 ± 0.01, β : 6.63± 0.01) were measured for the third deprotonation step involving the ellagic acid group, in good agreement with a prior literature report. The punicalagin anomer equilibrium readjusts in parallel with the proton dissociation steps as the pH is raised such that β-punicalagin becomes the most abundant anomer at neutral pH. The unusual upfield shifts observed for the glucose H3 and H5 resonances with increasing pH along with the shift in the α/β anomer equilibrium are likely the consequence of a conformational rearrangement.

  12. How Interfaces Affect the Acidity of the Anilinium Ion.

    PubMed

    Sripradite, Jarukorn; Miller, Susannah A; Johnson, Michael D; Tongraar, Anan; Crans, Debbie C

    2016-03-01

    The acidity of a compound is a fundamental property that dictates molecular speciation and reactivity in solution. Measurements of acidity of simple molecules in interfacial environments are rarely carried out but assumptions often are made that the difference is sufficiently small that the change can be ignored. The effect of oil-surfactant-water interfaces in reverse micellar systems on the pKa value of the anilinium ion was measured using titrations by NMR spectroscopy as the size of the bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane reverse micelles decreased. The pKa was observed to drop from 4.85±0.02 to 4.62±0.02 in water as the reverse micelle decreased from w(0) 10 to 4 (that is down to a reverse micellar radius of about 2 nm). NOSEY experiments demonstrated that the aniline moiety resides within the surfactant interface with the amine/ammonium moiety protruding into the waterpool bridging the interface. The presence of the aniline was found to have modest and variable effect on the size of the reverse micelles as observed using dynamic light scattering. Our experimental results provide information important to theoretical studies, which explore interface phenomena and provide a framework for information on such simple molecules. These studies quantitate the small but significant effect on the pKa values upon placement of an aromatic amine molecule at a hydrophilic-hydrophobic interface. PMID:26878992

  13. Redox potentials and pKa for benzoquinone from density functional theory based molecular dynamics.

    PubMed

    Cheng, Jun; Sulpizi, Marialore; Sprik, Michiel

    2009-10-21

    The density functional theory based molecular dynamics (DFTMD) method for the computation of redox free energies presented in previous publications and the more recent modification for computation of acidity constants are reviewed. The method uses a half reaction scheme based on reversible insertion/removal of electrons and protons. The proton insertion is assisted by restraining potentials acting as chaperones. The procedure for relating the calculated deprotonation free energies to Brønsted acidities (pK(a)) and the oxidation free energies to electrode potentials with respect to the normal hydrogen electrode is discussed in some detail. The method is validated in an application to the reduction of aqueous 1,4-benzoquinone. The conversion of hydroquinone to quinone can take place via a number of alternative pathways consisting of combinations of acid dissociations, oxidations, or dehydrogenations. The free energy changes of all elementary steps (ten in total) are computed. The accuracy of the calculations is assessed by comparing the energies of different pathways for the same reaction (Hess's law) and by comparison to experiment. This two-sided test enables us to separate the errors related with the restrictions on length and time scales accessible to DFTMD from the errors introduced by the DFT approximation. It is found that the DFT approximation is the main source of error for oxidation free energies. PMID:20568869

  14. Acid dissociation constants of uridine-5'-diphosphate compounds determined by 31phosphorus nuclear magnetic resonance spectroscopy and internal pH referencing.

    PubMed

    Jancan, Igor; Macnaughtan, Megan A

    2012-10-24

    The acid dissociation constant (pK(a)) of small, biological molecules is an important physical property used for investigating enzyme mechanisms and inhibitor design. For phosphorus-containing molecules, the (31)P nuclear magnetic resonance (NMR) chemical shift is sensitive to the local chemical environment, particularly to changes in the electronic state of the molecule. Taking advantage of this property, we present a (31)P NMR approach that uses inorganic phosphate buffer as an internal pH reference to determine the pK(a) values of the imide and second diphosphate of uridine-5'-diphosphate compounds, including the first reported values for UDP-GlcNAc and UDP-S-GlcNAc. New methods for using inorganic phosphate buffer as an internal pH reference, involving mathematical correction factors and careful control of the chemical shift reference sample, are illustrated. A comparison of the newly determined imide and diphosphate pK(a) values of UDP, UDP-GlcNAc, and UDP-S-GlcNAc with other nucleotide phosphate and thio-analogs reveals the significance of the monosaccharide and sulfur position on the pK(a) values.

  15. PKA catalytic subunit mutations in adrenocortical Cushing's adenoma impair association with the regulatory subunit.

    PubMed

    Calebiro, Davide; Hannawacker, Annette; Lyga, Sandra; Bathon, Kerstin; Zabel, Ulrike; Ronchi, Cristina; Beuschlein, Felix; Reincke, Martin; Lorenz, Kristina; Allolio, Bruno; Kisker, Caroline; Fassnacht, Martin; Lohse, Martin J

    2014-01-01

    We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA. PMID:25477193

  16. Calculated pKa Variations Expose Dynamic Allosteric Communication Networks.

    PubMed

    Lang, Eric J M; Heyes, Logan C; Jameson, Geoffrey B; Parker, Emily J

    2016-02-17

    Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

  17. Recent Advances in the Imaging Diagnosis of Hepatocellular Carcinoma: Value of Gadoxetic Acid-Enhanced MRI

    PubMed Central

    Joo, Ijin; Lee, Jeong Min

    2016-01-01

    Magnetic resonance imaging (MRI) using gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DPTA), or gadoxetic acid for short, is a hepatocyte-specific contrast agent which is now increasingly used for the detection and characterization of focal hepatic lesions, particularly in patients at high-risk of developing hepatocellular carcinomas (HCC). In fact, several recent guidelines now recognize gadoxetic acid-enhanced MRI (Gd-EOB-MRI) as the primary diagnostic imaging modality for the noninvasive diagnosis of HCC, although it must be noted that several major guidelines still include only extracellular contrast media-enhanced computed tomography and MRI. The primary merits of Gd-EOB-MRI lie in the fact that it can provide not only dynamic imaging, but also hepatobiliary phase (HBP) imaging which can lead to high lesion-to-liver contrast and give additional information regarding hepatocyte uptake via organic anion transporting polypeptides. This, in turn, allows higher sensitivity in detecting small HCCs and helps provide additional information regarding the multistep process of hepatocarcinogenesis. Indeed, many recent studies have investigated the diagnostic value of Gd-EOB-MRI for early HCCs as well as its role as a potential imaging biomarker in predicting outcome. We herein review the recent advances in the imaging diagnosis of HCCs focusing on the applications of Gd-EOB-MRI and the challenging issues that remain. PMID:26989660

  18. Recent Advances in the Imaging Diagnosis of Hepatocellular Carcinoma: Value of Gadoxetic Acid-Enhanced MRI.

    PubMed

    Joo, Ijin; Lee, Jeong Min

    2016-02-01

    Magnetic resonance imaging (MRI) using gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DPTA), or gadoxetic acid for short, is a hepatocyte-specific contrast agent which is now increasingly used for the detection and characterization of focal hepatic lesions, particularly in patients at high-risk of developing hepatocellular carcinomas (HCC). In fact, several recent guidelines now recognize gadoxetic acid-enhanced MRI (Gd-EOB-MRI) as the primary diagnostic imaging modality for the noninvasive diagnosis of HCC, although it must be noted that several major guidelines still include only extracellular contrast media-enhanced computed tomography and MRI. The primary merits of Gd-EOB-MRI lie in the fact that it can provide not only dynamic imaging, but also hepatobiliary phase (HBP) imaging which can lead to high lesion-to-liver contrast and give additional information regarding hepatocyte uptake via organic anion transporting polypeptides. This, in turn, allows higher sensitivity in detecting small HCCs and helps provide additional information regarding the multistep process of hepatocarcinogenesis. Indeed, many recent studies have investigated the diagnostic value of Gd-EOB-MRI for early HCCs as well as its role as a potential imaging biomarker in predicting outcome. We herein review the recent advances in the imaging diagnosis of HCCs focusing on the applications of Gd-EOB-MRI and the challenging issues that remain. PMID:26989660

  19. Lipid nutritional value of legumes: Evaluation of different extraction methods and determination of fatty acid composition.

    PubMed

    Caprioli, Giovanni; Giusti, Federica; Ballini, Roberto; Sagratini, Gianni; Vila-Donat, Pilar; Vittori, Sauro; Fiorini, Dennis

    2016-02-01

    This study sought to contribute to the assessment of the nutritional properties of legumes by determining the fatty acid (FA) composition of 29 legume samples after the evaluation of nine extraction methods. The Folch method and liquid-solid extraction with hexane/isopropanol or with hexane/acetone were investigated, as was the effect of previous hydration of samples. Soxhlet extractions were also evaluated with different solvent mixtures. Results on FA composition using the hexane/isopropanol extraction method were the same in terms of FA composition of the Folch method, but the extraction yield was only around 20-40% of that of the Folch method preceded by hydration. Some types of legumes showed particularly interesting values for the ratio of polyunsaturated fatty acids (PUFAs) n-6/n-3, such as lentils, with the value of 4.0, and Azuki beans, at 3.2. In lentils, the PUFAs% ranged from 42.0% to 57.4%, while in Azuki beans it was 57.5%. PMID:26304436

  20. Characterization and diagenesis of strong-acid carboxyl groups in humic substances

    USGS Publications Warehouse

    Leenheer, J.A.; Wershaw, R. L.; Brown, G.K.; Reddy, M.M.

    2003-01-01

    A small fraction of carboxylic acid functional groups in humic substances are exceptionally acidic with pKa values as low as 0.5. A review of acid-group theory eliminated most models and explanations for these exceptionally acidic carboxyl groups. These acidic carboxyl groups in Suwannee River fulvic acid were enriched by a 2-stage fractionation process and the fractions were characterized by elemental, molecular-weight, and titrimetric analyses, and by infrared and 13C- and 1H-nuclear magnetic resonance spectrometry. An average structural model of the most acidic fraction derived from the characterization data indicated a high density of carboxyl groups clustered on oxygen-heterocycle alicyclic rings. Intramolecular H-bonding between adjacent carboxyl groups in these ring structures enhanced stabilization of the carboxylate anion which results in low pKa1 values. The standard, tetrahydrofuran tetracarboxylic acid, was shown to have similar acidity characteristics to the highly acidic fulvic acid fraction. The end products of 3 known diagenetic pathways for the formation of humic substances were shown to result in carboxyl groups clustered on oxygen-heterocycle alicyclic rings.

  1. Effect of serum uric acid on the positive predictive value of dobutamine stress echocardiography.

    PubMed

    Aribas, Alpay; Akilli, Hakan; Kayrak, Mehmet; Alibasic, Hayrudin; Yildirim, Oguzhan; Sertdemir, Ahmet Lutfi; Karanfil, Mustafa; Ozdemir, Kurtulus

    2016-03-01

    There is controversial data regarding the relationship between uric acid (UA) and coronary artery disease and cardiovascular events. Despite the deleterious effects of hyperuricemia on endothelial function, the effect of UA on myocardial ischemia has not been previously studied. We aimed to investigate the relationship between UA and myocardial ischemia that was identified using dobutamine stress echocardiography (DSE). In this retrospective study, the laboratory and DSE reports of 548 patients were reviewed. The patients were divided into two groups based on the presence of ischemia and further subdivided into three groups according to the extent of ischemia (none, ischemia in 1-3 segments, ischemia in >3 segments). Serum UA levels were compared. Determinants of ischemia were assessed using a regression model. UA was increased in patients with ischemia and was correlated with the number of ischemic segments (p < 0.001). A cutoff value of UA > 5 mg/dl had 63.9 % sensitivity, 62.0 % specificity, 42.5 % positive predictive value (PPV), and 79.6 % negative predictive value for ischemia. When the positive DSE exams were further sorted according to the UA cutoff, the PPV of DSE increased from 80.2 to 94.0 %. Uric acid (odds ratio 1.51; 95 % CI 1.14-1.99), diabetes mellitus, HDL and glomerular filtration rate were found to be independent determinants of myocardial ischemia in DSE. Increased UA is associated with both the presence and extent of DSE-identified myocardial ischemia. A UA cutoff may be a good method to improve the PPV of DSE.

  2. Usnic acid controls the acidity tolerance of lichens.

    PubMed

    Hauck, Markus; Jürgens, Sascha-René

    2008-11-01

    The hypotheses were tested that, firstly, lichens producing the dibenzofuran usnic acid colonize substrates characterized by specific pH ranges, secondly, this preferred pH is in a range where soluble usnic acid and its corresponding anion occur in similar concentrations, and thirdly, usnic acid makes lichens vulnerable to acidity. Lichens with usnic acid prefer an ambient pH range between 3.5 and 5.5 with an optimum between 4.0 and 4.5. This optimum is close to the pK(a1) value of usnic acid of 4.4. Below this optimum pH, dissolved SO(2) reduces the chlorophyll fluorescence yield more in lichens with than without their natural content of usnic acid. This suggests that usnic acid influences the acidity tolerance of lichens. The putative mechanism of the limited acidity tolerance of usnic acid-containing lichens is the acidification of the cytosol by molecules of protonated usnic acid shuttling protons through the plasma membrane at an apoplastic pH

  3. Aqueous acidities of primary benzenesulfonamides: Quantum chemical predictions based on density functional theory and SMD.

    PubMed

    Aidas, Kęstutis; Lanevskij, Kiril; Kubilius, Rytis; Juška, Liutauras; Petkevičius, Daumantas; Japertas, Pranas

    2015-11-01

    Aqueous pK(a) of selected primary benzenesulfonamides are predicted in a systematic manner using density functional theory methods and the SMD solvent model together with direct and proton exchange thermodynamic cycles. Some test calculations were also performed using high-level composite CBS-QB3 approach. The direct scheme generally does not yield a satisfactory agreement between calculated and measured acidities due to a severe overestimation of the Gibbs free energy changes of the gas-phase deprotonation reaction by the used exchange-correlation functionals. The relative pK(a) values calculated using proton exchange method compare to experimental data very well in both qualitative and quantitative terms, with a mean absolute error of about 0.4 pK(a) units. To achieve this accuracy, we find it mandatory to perform geometry optimization of the neutral and anionic species in the gas and solution phases separately, because different conformations are stabilized in these two cases. We have attempted to evaluate the effect of the conformer-averaged free energies in the pK(a) predictions, and the general conclusion is that this procedure is highly too costly as compared with the very small improvement we have gained.

  4. PKA and PKC Are Required for Long-Term but Not Short-Term in Vivo Operant Memory in "Aplysia"

    ERIC Educational Resources Information Center

    Michel, Maximilian; Green, Charity L.; Lyons, Lisa C.

    2011-01-01

    We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in "Aplysia", learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term…

  5. Total Acid Value Titration of Hydrotreated Biomass Fast Pyrolysis Oil: Determination of Carboxylic Acids and Phenolics with Multiple End-Point Detection

    SciTech Connect

    Christensen, E.; Alleman, T. L.; McCormick, R. L.

    2013-01-01

    Total acid value titration has long been used to estimate corrosive potential of petroleum crude oil and fuel oil products. The method commonly used for this measurement, ASTM D664, utilizes KOH in isopropanol as the titrant with potentiometric end point determination by pH sensing electrode and Ag/AgCl reference electrode with LiCl electrolyte. A natural application of the D664 method is titration of pyrolysis-derived bio-oil, which is a candidate for refinery upgrading to produce drop in fuels. Determining the total acid value of pyrolysis derived bio-oil has proven challenging and not necessarily amenable to the methodology employed for petroleum products due to the different nature of acids present. We presented an acid value titration for bio-oil products in our previous publication which also utilizes potentiometry using tetrabutylammonium hydroxide in place of KOH as the titrant and tetraethylammonium bromide in place of LiCl as the reference electrolyte to improve the detection of these types of acids. This method was shown to detect numerous end points in samples of bio-oil that were not detected by D664. These end points were attributed to carboxylic acids and phenolics based on the results of HPLC and GC-MS studies. Additional work has led to refinement of the method and it has been established that both carboxylic acids and phenolics can be determined accurately. Use of pH buffer calibration to determine half-neutralization potentials of acids in conjunction with the analysis of model compounds has allowed us to conclude that this titration method is suitable for the determination of total acid value of pyrolysis oil and can be used to differentiate and quantify weak acid species. The measurement of phenolics in bio-oil is subject to a relatively high limit of detection, which may limit the utility of titrimetric methodology for characterizing the acidic potential of pyrolysis oil and products.

  6. LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

    PubMed

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  7. Value of Coproduction of Ethanol and Furfural from Acid Hydrolysis Processes

    SciTech Connect

    Parker, S.; Calnon, M.; Feinberg, D.; Power, A.; Weiss, L.

    1984-05-01

    In the acid hydrolysis of a cellulosic feedstock (wood, wood wastes, or crop residues), up to 3.65 lb of furfural may be coproduced with each gallon of ethanol for only the cost of recovering and purifying it. Each plant producing 50 x 106 gal/yr of ethanol would produce an amount of by-product furfural equal to the total current domestic production. Thus, the need arises for investigation into potentially suitable processes for deriving profitable end products from furfural and thus expanding the market. The objectives of this study were to determine the economic potential of five selected, large volume derivatives of furfural that could displace hydrocarbon-based chemicals, and the consequent value of furfural as a by-product to the cellulose hydrolysis process of ethanol production.

  8. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling.

    PubMed

    Bachmann, Verena A; Mayrhofer, Johanna E; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S; Stefan, Eduard

    2016-07-12

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein-protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein-protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions.

  9. Gpr161 anchoring of PKA consolidates GPCR and cAMP signaling

    PubMed Central

    Bachmann, Verena A.; Mayrhofer, Johanna E.; Ilouz, Ronit; Tschaikner, Philipp; Raffeiner, Philipp; Röck, Ruth; Courcelles, Mathieu; Apelt, Federico; Lu, Tsan-Wen; Baillie, George S.; Thibault, Pierre; Aanstad, Pia; Stelzl, Ulrich; Taylor, Susan S.; Stefan, Eduard

    2016-01-01

    Scaffolding proteins organize the information flow from activated G protein-coupled receptors (GPCRs) to intracellular effector cascades both spatially and temporally. By this means, signaling scaffolds, such as A-kinase anchoring proteins (AKAPs), compartmentalize kinase activity and ensure substrate selectivity. Using a phosphoproteomics approach we identified a physical and functional connection between protein kinase A (PKA) and Gpr161 (an orphan GPCR) signaling. We show that Gpr161 functions as a selective high-affinity AKAP for type I PKA regulatory subunits (RI). Using cell-based reporters to map protein–protein interactions, we discovered that RI binds directly and selectively to a hydrophobic protein–protein interaction interface in the cytoplasmic carboxyl-terminal tail of Gpr161. Furthermore, our data demonstrate that a binary complex between Gpr161 and RI promotes the compartmentalization of Gpr161 to the plasma membrane. Moreover, we show that Gpr161, functioning as an AKAP, recruits PKA RI to primary cilia in zebrafish embryos. We also show that Gpr161 is a target of PKA phosphorylation, and that mutation of the PKA phosphorylation site affects ciliary receptor localization. Thus, we propose that Gpr161 is itself an AKAP and that the cAMP-sensing Gpr161:PKA complex acts as cilium-compartmentalized signalosome, a concept that now needs to be considered in the analyzing, interpreting, and pharmaceutical targeting of PKA-associated functions. PMID:27357676

  10. Isoform-Selective Disruption of AKAP-Localized PKA Using Hydrocarbon Stapled Peptides

    PubMed Central

    2015-01-01

    A-kinase anchoring proteins (AKAPs) play an important role in the spatial and temporal regulation of protein kinase A (PKA) by scaffolding critical intracellular signaling complexes. Here we report the design of conformationally constrained peptides that disrupt interactions between PKA and AKAPs in an isoform-selective manner. Peptides derived from the A Kinase Binding (AKB) domain of several AKAPs were chemically modified to contain an all-hydrocarbon staple and target the docking/dimerization domain of PKA-R, thereby occluding AKAP interactions. The peptides are cell-permeable against diverse human cell lines, are highly isoform-selective for PKA-RII, and can effectively inhibit interactions between AKAPs and PKA-RII in intact cells. These peptides can be applied as useful reagents in cell-based studies to selectively disrupt AKAP-localized PKA-RII activity and block AKAP signaling complexes. In summary, the novel hydrocarbon-stapled peptides developed in this study represent a new class of AKAP disruptors to study compartmentalized RII-regulated PKA signaling in cells. PMID:24422448

  11. Hydrometallurgical recovery of metal values from sulfuric acid leaching liquor of spent lithium-ion batteries

    SciTech Connect

    Chen, Xiangping; Chen, Yongbin; Zhou, Tao Liu, Depei; Hu, Hang; Fan, Shaoyun

    2015-04-15

    Highlights: • Selective precipitation and solvent extraction were adopted. • Nickel, cobalt and lithium were selectively precipitated. • Co-D2EHPA was employed as high-efficiency extraction reagent for manganese. • High recovery percentages could be achieved for all metal values. - Abstract: Environmentally hazardous substances contained in spent Li-ion batteries, such as heavy metals and nocuous organics, will pose a threat to the environment and human health. On the other hand, the sustainable recycling of spent lithium-ion batteries may bring about environmental and economic benefits. In this study, a hydrometallurgical process was adopted for the comprehensive recovery of nickel, manganese, cobalt and lithium from sulfuric acid leaching liquor from waste cathode materials of spent lithium-ion batteries. First, nickel ions were selectively precipitated and recovered using dimethylglyoxime reagent. Recycled dimethylglyoxime could be re-used as precipitant for nickel and revealed similar precipitation performance compared with fresh dimethylglyoxime. Then the separation of manganese and cobalt was conducted by solvent extraction method using cobalt loaded D2EHPA. And McCabe–Thiele isotherm was employed for the prediction of the degree of separation and the number of extraction stages needed at specific experimental conditions. Finally, cobalt and lithium were sequentially precipitated and recovered as CoC{sub 2}O{sub 4}⋅2H{sub 2}O and Li{sub 2}CO{sub 3} using ammonium oxalate solution and saturated sodium carbonate solution, respectively. Recovery efficiencies could be attained as follows: 98.7% for Ni; 97.1% for Mn, 98.2% for Co and 81.0% for Li under optimized experimental conditions. This hydrometallurgical process may promise a candidate for the effective separation and recovery of metal values from the sulfuric acid leaching liquor.

  12. Hydrometallurgical recovery of metal values from sulfuric acid leaching liquor of spent lithium-ion batteries.

    PubMed

    Chen, Xiangping; Chen, Yongbin; Zhou, Tao; Liu, Depei; Hu, Hang; Fan, Shaoyun

    2015-04-01

    Environmentally hazardous substances contained in spent Li-ion batteries, such as heavy metals and nocuous organics, will pose a threat to the environment and human health. On the other hand, the sustainable recycling of spent lithium-ion batteries may bring about environmental and economic benefits. In this study, a hydrometallurgical process was adopted for the comprehensive recovery of nickel, manganese, cobalt and lithium from sulfuric acid leaching liquor from waste cathode materials of spent lithium-ion batteries. First, nickel ions were selectively precipitated and recovered using dimethylglyoxime reagent. Recycled dimethylglyoxime could be re-used as precipitant for nickel and revealed similar precipitation performance compared with fresh dimethylglyoxime. Then the separation of manganese and cobalt was conducted by solvent extraction method using cobalt loaded D2EHPA. And McCabe-Thiele isotherm was employed for the prediction of the degree of separation and the number of extraction stages needed at specific experimental conditions. Finally, cobalt and lithium were sequentially precipitated and recovered as CoC2O4 ⋅ 2H2O and Li2CO3 using ammonium oxalate solution and saturated sodium carbonate solution, respectively. Recovery efficiencies could be attained as follows: 98.7% for Ni; 97.1% for Mn, 98.2% for Co and 81.0% for Li under optimized experimental conditions. This hydrometallurgical process may promise a candidate for the effective separation and recovery of metal values from the sulfuric acid leaching liquor.

  13. Hydrometallurgical recovery of metal values from sulfuric acid leaching liquor of spent lithium-ion batteries.

    PubMed

    Chen, Xiangping; Chen, Yongbin; Zhou, Tao; Liu, Depei; Hu, Hang; Fan, Shaoyun

    2015-04-01

    Environmentally hazardous substances contained in spent Li-ion batteries, such as heavy metals and nocuous organics, will pose a threat to the environment and human health. On the other hand, the sustainable recycling of spent lithium-ion batteries may bring about environmental and economic benefits. In this study, a hydrometallurgical process was adopted for the comprehensive recovery of nickel, manganese, cobalt and lithium from sulfuric acid leaching liquor from waste cathode materials of spent lithium-ion batteries. First, nickel ions were selectively precipitated and recovered using dimethylglyoxime reagent. Recycled dimethylglyoxime could be re-used as precipitant for nickel and revealed similar precipitation performance compared with fresh dimethylglyoxime. Then the separation of manganese and cobalt was conducted by solvent extraction method using cobalt loaded D2EHPA. And McCabe-Thiele isotherm was employed for the prediction of the degree of separation and the number of extraction stages needed at specific experimental conditions. Finally, cobalt and lithium were sequentially precipitated and recovered as CoC2O4 ⋅ 2H2O and Li2CO3 using ammonium oxalate solution and saturated sodium carbonate solution, respectively. Recovery efficiencies could be attained as follows: 98.7% for Ni; 97.1% for Mn, 98.2% for Co and 81.0% for Li under optimized experimental conditions. This hydrometallurgical process may promise a candidate for the effective separation and recovery of metal values from the sulfuric acid leaching liquor. PMID:25619126

  14. 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.

  15. A color-determining amino acid residue of proteorhodopsin.

    PubMed

    Ozaki, Yuya; Kawashima, Takayoshi; Abe-Yoshizumi, Rei; Kandori, Hideki

    2014-09-30

    Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria. More than 1000 PRs are classified as blue-absorbing (λmax ∼ 490 nm) and green-absorbing (λmax ∼ 525 nm) PRs. The color determinant is known to be at position 105, where blue-absorbing and green-absorbing PRs possess Gln and Leu, respectively. This suggests hydrophobicity at position 105 plays a key role in color tuning. Here we successfully introduced 19 amino acid residues into position 105 of green-absorbing PR in the membrane environment and investigated the absorption properties. High-performance liquid chromatography analysis shows that the isomeric composition of the all-trans form is >70% for all mutants, indicating little influence of different isomers on color tuning. Absorption spectra of the wild-type and 19 mutant proteins were well-characterized by the pH-dependent equilibria of the protonated and deprotonated counterion (Asp97) of the Schiff base, whereas the λmax values of these two states and the pKa value differed significantly among mutants. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, the λmax values of the two states and the pKa value did not correlate with the hydropathy index of residues. In contrast, the λmax and pKa were correlated with the volume of residues, though Gln and Leu possess similar volumes. This observation concludes that the λmax and pKa of Asp97 are determined by local and specific interactions in the Schiff base moiety, in which the volume of the residue at position 105 is more influential than its hydrophobicity. We suggest that the hydrogen-bonding network in the Schiff base moiety plays a key role in the λmax and pKa of Asp97, and the hydrogen-bonding network is significantly perturbed by large amino acid residues but may be preserved by additional water molecule(s) for small amino acid residues at position 105. PMID:25180875

  16. Fundamental thermochemical properties of amino acids: gas-phase and aqueous acidities and gas-phase heats of formation.

    PubMed

    Stover, Michele L; Jackson, Virgil E; Matus, Myrna H; Adams, Margaret A; Cassady, Carolyn J; Dixon, David A

    2012-03-01

    The gas-phase acidities of the 20 L-amino acids have been predicted at the composite G3(MP2) level. A broad range of structures of the neutral and anion were studied to determine the lowest energy conformer. Excellent agreement is found with the available experimental gas-phase deprotonation enthalpies, and the calculated values are within experimental error. We predict that tyrosine is deprotonated at the CO(2)H site. Cysteine is predicted to be deprotonated at the SH but the proton on the CO(2)H is shared with the S(-) site. Self-consistent reaction field (SCRF) calculations with the COSMO parametrization were used to predict the pK(a)'s of the non-zwitterion form in aqueous solution. The differences in the non-zwitterion pK(a) values were used to estimate the free energy difference between the zwitterion and nonzwitterion forms in solution. The heats of formation of the neutral compounds were calculated from atomization energies and isodesmic reactions to provide the first reliable set of these values in the gas phase. Further calculations were performed on five rare amino acids to predict their heats of formation, acidities, and pK(a) values.

  17. Acid-base properties of the nucleic-acid model 2'-deoxyguanylyl(5'-->3')-2'-deoxy-5'-guanylate, d(pGpG)3-, and of related guanine derivatives.

    PubMed

    Knobloch, Bernd; Sigel, Helmut; Okruszek, Andrzej; Sigel, Roland K O

    2006-03-21

    The dinucleotide d(pGpG) is an often employed DNA model to study various kinds of interactions between DNA and metal ions, but its acid-base properties were not yet described in detail. In this study the six deprotonation reactions of H4[d(pGpG)]+ are quantified. The acidity constants for the release of the first proton from the terminal P(O)(OH)2 group (pKa = 0.65) and for one of the (N7)H+ sites (pKa = 2.4) are estimated. The acidity constants of the remaining four deprotonation reactions were measured by potentiometric pH titrations in aqueous solution (25 degrees C; I = 0.1 M, NaNO3): The pKa values for the deprotonations of the second (N7)H+, the P(O)2(OH)-, and the two (N1)H sites are 2.98, 6.56, 9.54 and 10.11, respectively. Based on these results we show how to estimate acidity constants for related systems that have not been studied, e.g. pGpG, which is involved in the initiation step of a rotavirus RNA polymerase. The relevance of our results for nucleic acids in general is briefly indicated.

  18. Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation.

    PubMed

    Li, Jiaxin; Dou, Xiaobing; Li, Songtao; Zhang, Ximei; Zeng, Yong; Song, Zhenyuan

    2015-11-01

    Nicotinamide (NAM) is the amide of nicotinic acid and a predominant precursor for NAD(+) biosynthesis via the salvage pathway. Sirt1 is a NAD(+)-dependent deacetylase, playing an important role in regulating cellular functions. Although hepatoprotective effect of NAM has been reported, the underlying mechanism remains elusive. ER stress, induced by saturated fatty acids, in specific palmitate, plays a pathological role in the development of nonalcoholic fatty liver disease. This study aims to determine the effect of NAM on palmitate-induced ER stress in hepatocytes and to elucidate molecular mechanisms behind. Both HepG2 cells and primary mouse hepatocytes were exposed to palmitate (conjugated to BSA at a 2:1 M ratio), NAM, or their combination for different durations. Cellular NAD(+) level, Sirt1 expression/activity, ER stress, as well as cAMP/PKA/CREB pathway activation were determined. NAM increased Sirt1 expression and enzymatic activity, which contributes to the ameliorative effect of NAM on palmitate-triggered ER stress. NAM increased intracellular NAD(+) level in hepatocytes, however, blocking the salvage pathway, a pathway for NAD(+) synthesis from NAM, only partially prevented NAM-induced Sirt1 upregulation while completely prevented NAD+ increase in response to NAM. Further mechanistic investigations revealed that NAM elevated intracellular cAMP level via suppressing PDE activity, leading to downstream PKA and CREB activation. Importantly, cAMP/PKA/CREB pathway blockade abolished not only NAM-induced Sirt1 upregulation, but also its protective effect against ER stress. Our results demonstrate that NAM protects hepatocytes against palmitate-induced ER stress in hepatocytes via upregulating Sirt1. Activation of the cAMP/PKA/CREB pathway plays a key role in NAM-induced Sirt1 upregulation. PMID:26352206

  19. Molecular cloning, genomic organization, chromosomal mapping and subcellular localization of mouse PAP7: a PBR and PKA-RIalpha associated protein.

    PubMed

    Liu, Jun; Cavalli, Luciane R; Haddad, Bassem R; Papadopoulos, Vassilios

    2003-04-10

    A mouse protein that interacts with the peripheral-type benzodiazepine receptor (PBR) and the cAMP-dependent protein kinase A (PKA) regulatory subunit RIalpha (PKA-RIalpha), named PBR and PKA associated protein 7 (PAP7) was identified and shown to be involved in hormone-induced steroid biosynthesis in testicular Leydig cells. In the present study, mouse PAP7 cDNA was extended by 5'-rapid amplification of cDNA ends; and a 3432 bp sequence, encoding a 525-amino-acid protein with a calculated molecular weight of 60 kDa, was re-assembled. Mouse and human PAP7 share an 85% amino acid identity and contain a conserved acyl-CoA-binding protein/diazepam binding inhibitor (ACBP/DBI) motif. ACBP/DBI has been identified as the endogenous PBR ligand able to stimulate mitochondrial steroid formation in all steroidogenic cells. The full-length mouse PAP7 gene was cloned and assembled by screening a BAC clone, polymerase chain reaction and searching the mouse genome database. The gene is approximately 29 kb in length and includes eight exons and seven introns. Although it is shorter than the human PAP7 gene, all exons are conserved between the mouse and human. The mouse PAP7 gene was mapped to chromosome 1H3-5 by fluorescence in situ hybridization in agreement with in silico search of the mouse genome database that mapped the PAP7 cDNA sequence to the 1H4 area. Immunofluorescence confocal microscopy demonstrated that PAP7 is mainly localized in the trans-Golgi apparatus and mitochondria in mouse tumor Leydig cells, in agreement with its proposed function in targeting the PKA isoenzyme to organelles rich in PBR, i.e. mitochondria, where phosphorylation of specific protein substrates mediates the hormone-induced steroid formation.

  20. Targeting cAMP/PKA pathway for glycemic control and type 2 diabetes therapy.

    PubMed

    Yang, Haihua; Yang, Linghai

    2016-08-01

    In mammals, cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that is usually elicited by binding of hormones and neurotransmitters to G protein-coupled receptors (GPCRs). cAMP exerts many of its physiological effects by activating cAMP-dependent protein kinase (PKA), which in turn phosphorylates and regulates the functions of downstream protein targets including ion channels, enzymes, and transcription factors. cAMP/PKA signaling pathway regulates glucose homeostasis at multiple levels including insulin and glucagon secretion, glucose uptake, glycogen synthesis and breakdown, gluconeogenesis, and neural control of glucose homeostasis. This review summarizes recent genetic and pharmacological studies concerning the regulation of glucose homeostasis by cAMP/PKA in pancreas, liver, skeletal muscle, adipose tissues, and brain. We also discuss the strategies for targeting cAMP/PKA pathway for research and potential therapeutic treatment of type 2 diabetes mellitus (T2D). PMID:27194812

  1. PKA Knockdown Enhances Cell Killing In Response To Radiation And Androgen Deprivation

    PubMed Central

    Hensley, Harvey H.; Hannoun-Levi, Jean-Michel; Hachem, Paul; Mu, Zhaomei; Stoyanova, Radka; Khor, Li-Yan; Agrawal, Sudhir; Pollack, Alan

    2010-01-01

    The therapeutic efficacy of Gem®231, a second generation antisense molecule targeted to the RIα subunit of PKARIα (AS-PKA), administered in combination with androgen deprivation (AD) and radiation therapy (RT), was examined in androgen sensitive (LNCaP) and insensitive (PC3) cell lines. Apoptosis was assayed by Caspase 3+7 activity and AnnexinV binding. AS-PKA significantly increased apoptosis in vitro from RT (both lines), with further increases in LNCaP cells grown in AD medium. In LNCaP cells, AD increased phosphorylated map-kinase (pMAPK), which was reduced by AS-PKA relative to the MM controls. AS-PKA also reduced pMAPK levels in PC3 cells. Cell death was measured by clonogenic survival assays. In vivo, LNCaP cells were grown orthotopically in nude mice. Tumor kinetics were measured by magnetic resonance imaging and serum prostate-specific antigen. PC3 cells were grown subcutaneously and tumor volume assessed by caliper measurements. In PC3 xenografts, AS-PKA caused a significant increase in tumor doubling time relative to MM controls as a monotherapy or in combination with RT. In orthotopic LNCaP tumors, AS-PKA was ineffective as a monotherapy; however it caused a statistically significant increase in tumor doubling time relative to MM controls when used in combination with AD, with or without RT. PKARIα levels in tumors were quantified via immunohistochemical (IHC) staining and image analysis. IHC measurements in LNCaP cells showed that AS-PKA reduced PKARIα levels in vivo. We demonstrate for the first time that AS-PKA enhances cell killing androgen sensitive prostate cancer cells to AD±RT and androgen insensitive cells to RT. PMID:20960462

  2. Dynamic Protonation Equilibrium of Solvated Acetic Acid

    SciTech Connect

    Gu, Wei; Frigato, Tomaso; Straatsma, TP; Helms, Volkhard H.

    2007-04-13

    For the first time, the dynamic protonation equilibrium between an amino acid side chain analogue and bulk water as well as the diffusion properties of the excess proton were successfully reproduced through unbiased computer simulations. During a 50 ns Q-HOP MD simulation, two different regimes of proton transfer were observed. Extended phases of frequent proton swapping between acetic acid and nearby water were separated by phases where the proton freely diffuses in the simulation box until it is captured again by acetic acid. The pKa of acetic acid was calculated around 3.0 based on the relative population of protonated and deprotonated states and the diffusion coefficient of excess proton was computed from the average mean squared displacement in the simulation. Both calculated values agree well with the experimental measurements.

  3. PKA Enhances the Acute Insulin Response Leading to the Restoration of Glucose Control

    PubMed Central

    Kaihara, Kelly A.; Dickson, Lorna M.; Ellenbroek, Johanne H.; Orr, Caitlin M.D.; Layden, Brian T.

    2015-01-01

    Diabetes arises from insufficient insulin secretion and failure of the β-cell mass to persist and expand. These deficits can be treated with ligands to Gs-coupled G-protein-coupled receptors that raise β-cell cAMP. Here we studied the therapeutic potential of β-cell cAMP-dependent protein kinase (PKA) activity in restoring glucose control using β-caPKA mice. PKA activity enhanced the acute insulin response (AIR) to glucose, which is a primary determinant of the efficacy of glucose clearance. Enhanced AIR improved peripheral insulin action, leading to more rapid muscle glucose uptake. In the setting of pre-established glucose intolerance caused by diet-induced insulin resistance or streptozotocin-mediated β-cell mass depletion, PKA activation enhanced β-cell secretory function to restore glucose control, primarily through augmentation of the AIR. Enhanced AIR and improved glucose control were maintained through 16 weeks of a high-fat diet and aging to 1 year. Importantly, improved glucose tolerance did not increase the risk for hypoglycemia, nor did it rely upon hyperinsulinemia or β-cell hyperplasia, although PKA activity was protective for β-cell mass. These data highlight that improving β-cell function through the activation of PKA has a large and underappreciated capacity to restore glucose control with minimal risk for adverse side effects. PMID:25475437

  4. Interaction of PACAP with Sonic hedgehog reveals complex regulation of the hedgehog pathway by PKA.

    PubMed

    Niewiadomski, Pawel; Zhujiang, Annie; Youssef, Mary; Waschek, James A

    2013-11-01

    Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (cGCPs) and its aberrant activation causes a cerebellar cancer medulloblastoma. Pituitary adenylate cyclase activating polypeptide (PACAP) inhibits Shh-driven proliferation of cGCPs and acts as tumor suppressor in murine medulloblastoma. We show that PACAP blocks canonical Shh signaling by a mechanism that involves activation of protein kinase A (PKA) and inhibition of the translocation of the Shh-dependent transcription factor Gli2 into the primary cilium. PKA is shown to play an essential role in inhibiting gene transcription in the absence of Shh, but global PKA activity levels are found to be a poor predictor of the degree of Shh pathway activation. We propose that the core Shh pathway regulates a small compartmentalized pool of PKA in the vicinity of primary cilia. GPCRs that affect global PKA activity levels, such as the PACAP receptor, cooperate with the canonical Shh signal to regulate Gli protein phosphorylation by PKA. This interaction serves to fine-tune the transcriptional and physiological function of the Shh pathway. PMID:23872071

  5. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    SciTech Connect

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression.

  6. Acid titrations of poly(dG-dC).poly(dG-dC) in aqueous solution and in a w/o microemulsion.

    PubMed

    Airoldi, Marta; Boicelli, C Andrea; Gennaro, Guiseppe; Giomini, Marcello; Giuliani, Anna Maria; Giustini, Mauro

    2006-02-01

    The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.

  7. How acidic are monomeric structural units of heparin?

    NASA Astrophysics Data System (ADS)

    Remko, Milan; Broer, Ria; Van Duijnen, Piet Th.

    2013-12-01

    Density functional theory methods with the B3LYP functional have been used to letter the acidity of carboxyl, O-sulfo and N-sulfo groups in six basic monomeric structural units of heparin (1-OMe ΔUA-2S, 1-OMe GlcN-S6S, 1,4-DiOMe GlcA, 1,4-DiOMe GlcN-S3S6S, 1,4-DiOMe IdoA-2S, and 1,4-DiOMe GlcN-S6S). The predicted gas-phase acidity of the acidic functional groups in the monomeric structural units of heparin is: O-sulfo > N-sulfo > carboxyl. The computed pKa values provide the same order of acidity as was observed in water solution. This implies that hydration does not change ordering of acidity of major acidic groups of monomeric structural units of heparin.

  8. Substituent effects and pH profiles for stability constants of arylboronic acid diol esters.

    PubMed

    Martínez-Aguirre, Mayte A; Villamil-Ramos, Raul; Guerrero-Alvarez, Jorge A; Yatsimirsky, Anatoly K

    2013-05-17

    Stability constants of boronic acid diol esters in aqueous solution have been determined potentiometrically for a series of meta-, para-substituted phenylboronic acids and diols of variable acidity. The constants β(11-1) for reactions between neutral forms of reactants producing the anionic ester plus proton follow the Hammett equation with ρ depending on pKa of diol and varying from 2.0 for glucose to 1.29 for 4-nitrocatechol. Observed stability constants (K(obs)) measured by UV-vis and fluorometric titrations at variable pH for esters of 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron) generally agree with those expected on the basis of β(11-1) values, but the direct fitting of K(obs) vs pH profiles gives shifted pKa values both for boronic acids and diol as a result of significant interdependence of fitting parameters. The subsituent effects on absorption and fluorescence spectra of Tiron arylboronate esters are characterized. The K(obs) for Tiron determined by (11)B NMR titrations are approximately 1 order of magnitude smaller than those determined by UV-vis titrations under identical conditions. A general equation, which makes possible an estimate of β(11-1) for any pair of boronic acid and diol from their pKa values, is proposed on the basis of established Brönsted-type correlation of Hammett parameters for β(11-1) with acidity of diols. The equation allows one to calculate stability constants expected only on basis of acid-base properties of the components, thus permitting more strict evaluation of contributions of additional factors such as steric or charge effects to the ester stability.

  9. Fractionation of oil sands-process affected water using pH-dependent extractions: a study of dissociation constants for naphthenic acids species.

    PubMed

    Huang, Rongfu; Sun, Nian; Chelme-Ayala, Pamela; McPhedran, Kerry N; Changalov, Mohamed; Gamal El-Din, Mohamed

    2015-05-01

    The fractionation of oil sands process-affected water (OSPW) via pH-dependent extractions was performed to quantitatively investigate naphthenic acids (NAs, CnH2n+ZO2) and oxidized NAs (Ox-NAs) species (CnH2n+ZO3 and CnH2n+ZO4) using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOFMS). A mathematical model was also developed to estimate the dissociation constant pKa for NAs species, considering the liquid-liquid extraction process and the aqueous layer acid-base equilibrium. This model provides estimated dissociation constants for compounds in water samples based on fractionation extraction and relative quantification. Overall, the sum of O2-, O3-, and O4-NAs species accounted for 33.6% of total extracted organic matter. Accumulative extracted masses at different pHs revealed that every oxygen atom added to NAs increases the pKa (i.e., O2-NAspKa of O2-, O3-, and O4-NAs. The model obtained estimated pKa values of 3.5 for O2-NAs, 4.8 for O3-NAs, and 6.8 for O4-NAs via nonlinear regression curve fittings. These pKa values are valuable physicochemical parameters for environmental engineering applications targeting OSPW NAs treatment.

  10. Short communication: Effects of prepartum diets supplemented with rolled oilseeds on Brix values and fatty acid profile of colostrum.

    PubMed

    Salehi, R; Ambrose, D J; Oba, M

    2016-05-01

    The objective of this study was to evaluate effects of oilseeds supplemented in prepartum diets on colostrum quality. Thirty-nine dry pregnant Holstein cows (14 primiparous and 25 multiparous cows) were blocked by body condition score and parity and assigned to 1 of 3 experimental diets containing rolled oilseeds at 8% of dietary dry matter (canola seed or sunflower seed) or no oilseed (control) at 35 d before the expected calving date. Canola seed is high in oleic acid and sunflower seed is high in linoleic acid content. Colostrum samples were collected at the first milking after calving, and concentrations of nutrient composition, fatty acid profile, and Brix value (an indicator IgG concentration) were determined. Cows fed sunflower seeds before calving produced colostrum with greater crude protein content (15.0 vs. 12.9%), colostral Brix values (24.3 vs. 20.3%), and conjugated linoleic acid concentration (18:2 cis-9,trans-11; 0.64 vs. 0.48%) compared with those fed canola seed. Positive effects of feeding sunflower seed might be mediated by ruminal metabolism of linoleic acid and subsequent enhanced production of conjugated linoleic acid. Oilseed supplementation in prepartum diets of dairy cows also altered fatty acid profile of colostrum in a way to reflect fatty acid profile of the supplemented oilseeds except for oleic acid. In conclusion, prepartum feeding of sunflower seed increased colostral Brix value, an indicator of colostral IgG concentration, compared with that of canola seed, but its mode of action and effects on health and productivity of calves need to be investigated.

  11. Free amino acids in crocodilians fed proteins of different biological value.

    PubMed

    Herbert, J D; Coulson, R A

    1975-05-01

    Changes in plasma levels of amino acids derived from fed protein were determined by feeding crocodilians (Caiman crocodilus crocodilus and Alligator mississipiensis) 7.5 g protein/kg body weight and by monitoring the plasma free amino acids for several days. Zein and several other vegetable proteins produced no rise in plasma amino acids and were excreted intact in the feces. Casein and fish muscle were rapidly digested but produced little rise in plasma amino acids, and the increases showed no relationship to the composition of the protein fed. Gelatin feeding led to large increases in plasma amino acids that persisted for more than a week, and the resulting pattern was nearly identical to the composition of gelatin with the exception of aspartic and glutamic acids, and several animals died. Equivalent quantities of fish muscle protein were assimilated without difficulty by the crocodilians. Endogenous protein secreted into the gut apparently contributed little to the amino acid mixture absorbed.

  12. Nutritional value of protein hydrolysis products (oligopeptides and free amino acids) as a consequence of absorption and metabolism kinetics

    NASA Technical Reports Server (NTRS)

    Rerat, A.

    1995-01-01

    When pigs were submitted to duodenal infusion of solutions containing a large percentage of small peptides (PEP) or free amino acids with the same pattern (AAL) amino acids appear in the portal blood more rapidly and more uniformly after infusion of PEP then after infusion of AAL, with the notable exception of methionine for which the opposite was true. These differences were lowered when a carbohydrate (maltose dextrin) was present in the solution, but nevertheless remained significant for the first hour after the infusion. The long-term (8-hour) uptake of free amino acids into the liver and the peripheral tissues differed in profile according to the nature of the duodenal infusion. Peripheral uptake was appreciably less well balanced after infusion of free amino acids (deficiency of threonine and phenylalanine) than after infusion of small peptides (deficiency of methionine). Accordingly, in the rat, under conditions of discontinuous enteral nutrition the mixture of small peptides was of greater nutritive value than the mixture of free amino acids. It thus appears that the absorption kinetics which results in important variations in the temporal distribution of free amino acids in the tissues may be at the origin of transitory imbalances in tissue amino acid uptake, and as a result of a lower nutritive value.

  13. Photophysical, photochemical, and thermodynamic properties of shikimic acid derivatives: calycin and rhizocarpic acid (lichens).

    PubMed

    Hidalgo, M E; Fernández, E; Ponce, M; Rubio, C; Quilhot, W

    2002-04-01

    Photophysical and photochemical parameters of the lichen metabolites calycin and rhizocarpic acid were determined. Experiments were carried out in micellar solutions of 3% Brij 35, at pH 2 and 12, and in acetonitrile. Both metabolites absorb in the UV-A and UV-B regions, and emit fluorescence in the visible region of the solar spectrum. Shifts were not observed in the absorption spectra, at pH 2 and 12. The low phi(c), between 10(-5) and 10(-2), shows that both compounds are photostable in the experimental conditions. For rhizocarpic acid, two values of pK(a) were obtained: 5.1 corresponding to the hydroxyl group, and 9.0 corresponding to the protonated nitrogen. Calycin presents only one value of pK(a): 4.9, that is attributed to the hydroxyl group. L-(+)-Gluconic-gamma-lactonic acid was used as a reference model; the compound showed greater photoinstability, demonstrating that the photodegradation observed occurs mainly in the oxolane carbonylic ring.

  14. Opposing needling promotes behavior recovery and exerts neuroprotection via the cAMP/PKA/CREB signal transduction pathway in transient MCAO rats

    PubMed Central

    JIANG, YIJING; YANG, SHANLI; TAO, JING; LIN, ZHICHENG; YE, XIAOQIAN; YOU, YONGMEI; PENG, JUN; HONG, ZHENFENG; CHEN, LIDIAN

    2016-01-01

    The aim of the present study was to investigate whether the cyclic adenosine 3′,5′-monophosphate (cAMP)/protein kinase A(PKA)/cAMP-responsive element binding protein (CREB) signal transduction pathway triggered by γ-aminobutyric acid class B (GABAB) receptor activation is involved in neuroprotection against ischemia and behavioral recovery induced by opposing needling (ON). A total of 80 rats were randomly divided into four groups: A sham operation group, an ischemia group, an ON group and an ON group effectively inhibited by the GABAB receptor antagonist, CGP35384 (n=20/group). The behavior of the rats was assessed by their neurological deficit score, whereas the impairment of gait was examined using the CatWalk system. The volume of cerebral infarction was examined upon treatment with 2,3,5-triphenyltetrazolium chloride. The expression levels of CREB, GABAB1 and GABAB2 were examined by western blotting and reverse transcription-quantitative polymerase chain reaction, and the activity of adenylyl cyclase (AC), cAMP and PKA in the serum was detected using an enzyme-linked immunosorbent assay. In the present study, in comparison with other groups, the ON group exhibited a reduced score for the neurological deficit, the stride length and swing speed were improved, and the volume of infarction was reduced. However, these effects were reversed upon administration of CGP35384. Additionally, the expression levels of CREB, GABAB1 and GABAB2 were increased in the ON group. The levels of AC, cAMP and PKA in the serum were also increased in the ON group, whereas the addition of CGP35384 reversed these effects. The results of the present study demonstrated that ON markedly protected the brain against transient cerebral ischemic injury, and this effect was possibly mediated by the activation of the GABAB/cAMP/PKA/CREB signal transduction pathway. These findings implied that ON may be a potential therapeutic method for treating stroke. PMID:26780954

  15. Neuroprotective effect of vitamin C against the ethanol and nicotine modulation of GABA(B) receptor and PKA-alpha expression in prenatal rat brain.

    PubMed

    Naseer, M I; Lee, H Y; Kim, M O

    2010-06-01

    Prenatal ethanol exposure has various deleterious effects on neuronal development and can induce various defects in developing brain, resulting in fetal alcohol syndrome (FAS). gamma-Aminobutyric acid (GABA(B)) receptor (R) is known to play an important role during the development of the central nervous system (CNS). Our study was designed to investigate the effect of ethanol (100 mM), nicotine (50 microM) (for 30 min and 1 h), vitamin C (vitC, 0.5 mM), ethanol plus vitC, and nicotine plus vitC on expression level of GABA(B1), GABA(B2)R, and protein kinase A-alpha (PKA) in prenatal rat cortical and hippocampal neurons at gestational days (GD) 17.5. The results showed that, upon ethanol and nicotine exposure, GABA(B1) and GABA(B2)R protein expression increased significantly in the cortex and hippocampus for a short (30 min) and long term (1 h), whereas only GABA(B2)R subunit was decreased upon nicotine exposure for a long term in the cortex. Furthermore, PKA expression in cortex and hippocampus increased with ethanol exposure during short term, whereas long-term exposure results increased in cortex and decreased in hippocampus. Moreover, the cotreatment of vitC with ethanol and nicotine showed significantly decreased expression of GABA(B1), GABA(B2)R, and PKA in cortex and hippocampus for a long-term exposure. Mitochondrial membrane potential, Fluoro-jade-B, and propidium iodide staining were used to elucidate possible neurodegeneration. Our results suggest the involvement of GABA(B)R and PKA in nicotine and ethanol-mediated neurodevelopmental defects and the potential use of vitC as a effective protective agent for FAS-related deficits.

  16. Determination of the pKa of ionizable enzyme groups by nonlinear regression using a second degree equation.

    PubMed

    O'Reilly, S; Riveros, M C

    1994-01-01

    A second degree equation fitted by nonlinear regression for the analysis of the pH effect on enzyme activity is proposed for diprotic enzyme systems. This method allows the calculation of two molecular dissociation constants (KE1 and KE2 for the free enzyme, KES1 and KES2 for the ES complex) and the pH independent parameters (Vmax and Vmax/Km). The method is validated by bibliographic (alpha-chymotrypsin) and experimental data (almond beta-D-glucosidase). No significant differences were found between present data and those previously reported in the literature using similar experimental conditions. This method works using comparatively few [H+] concentration values within a narrow pH range, preferentially around the optimum, being adequate for diprotic systems with close pKa values. PMID:8728828

  17. Determination of the pKa of ionizable enzyme groups by nonlinear regression using a second degree equation.

    PubMed

    O'Reilly, S; Riveros, M C

    1994-01-01

    A second degree equation fitted by nonlinear regression for the analysis of the pH effect on enzyme activity is proposed for diprotic enzyme systems. This method allows the calculation of two molecular dissociation constants (KE1 and KE2 for the free enzyme, KES1 and KES2 for the ES complex) and the pH independent parameters (Vmax and Vmax/Km). The method is validated by bibliographic (alpha-chymotrypsin) and experimental data (almond beta-D-glucosidase). No significant differences were found between present data and those previously reported in the literature using similar experimental conditions. This method works using comparatively few [H+] concentration values within a narrow pH range, preferentially around the optimum, being adequate for diprotic systems with close pKa values.

  18. Studies of mice with cyclic AMP-dependent protein kinase (PKA) defects reveal the critical role of PKA's catalytic subunits in anxiety.

    PubMed

    Briassoulis, George; Keil, Margaret F; Naved, Bilal; Liu, Sophie; Starost, Matthew F; Nesterova, Maria; Gokarn, Nirmal; Batistatos, Anna; Wu, T John; Stratakis, Constantine A

    2016-07-01

    Cyclic adenosine mono-phosphate-dependent protein kinase (PKA) is critically involved in the regulation of behavioral responses. Previous studies showed that PKA's main regulatory subunit, R1α, is involved in anxiety-like behaviors. The purpose of this study was to determine how the catalytic subunit, Cα, might affect R1α's function and determine its effects on anxiety-related behaviors. The marble bury (MB) and elevated plus maze (EPM) tests were used to assess anxiety-like behavior and the hotplate test to assess nociception in wild type (WT) mouse, a Prkar1a heterozygote (Prkar1a(+/-)) mouse with haploinsufficiency for the regulatory subunit (R1α), a Prkaca heterozygote (Prkaca(+/-)) mouse with haploinsufficiency for the catalytic subunit (Cα), and a double heterozygote mouse (Prkar1a(+/-)/Prkaca(+/-)) with haploinsufficiency for both R1α and Cα. We then examined specific brain nuclei involved in anxiety. Results of MB test showed a genotype effect, with increased anxiety-like behavior in Prkar1a(+/-) and Prkar1a(+/-)/Prkaca(+/-) compared to WT mice. In the EPM, Prkar1a(+/-) spent significantly less time in the open arms, while Prkaca(+/-) and Prkar1a(+/-)/Prkaca(+/-) mice displayed less exploratory behavior compared to WT mice. The loss of one Prkar1a allele was associated with a significant increase in PKA activity in the basolateral (BLA) and central (CeA) amygdala and ventromedial hypothalamus (VMH) in both Prkar1a(+/-) and Prkar1a(+/-)/Prkaca(+/-) mice. Alterations of PKA activity induced by haploinsufficiency of its main regulatory or most important catalytic subunits result in anxiety-like behaviors. The BLA, CeA, and VMH are implicated in mediating these PKA effects in brain. PMID:26992826

  19. Arginine-82 regulates the pKa of the group responsible for the light-driven proton release in bacteriorhodopsin.

    PubMed Central

    Govindjee, R; Misra, S; Balashov, S P; Ebrey, T G; Crouch, R K; Menick, D R

    1996-01-01

    In wild-type bacteriorhodopsin light-induced proton release occurs before uptake at neutral pH. In contrast, in mutants in which R82 is replaced by a neutral residue (as in R82A and R82Q), only a small fraction of the protons is released before proton uptake at neutral pH; the major fraction is released after uptake. In R82Q the relative amounts of the two types of proton release, "early" (preceding proton uptake) and "late" (following proton uptake), are pH dependent. The main conclusions are that 1) R82 is not the normal light-driven proton release group; early proton release can be observed in the R82Q mutant at higher pH values, suggesting that the proton release group has not been eliminated. 2) R82 affects the pKa of the proton release group both in the unphotolyzed state of the pigment and during the photocycle. In the wild type (in 150 mM salt) the pKa of this group decreases from approximately 9.5 in the unphotolyzed pigment to approximately 5.8 in the M intermediate, leading to early proton release at neutral pH. In the R82 mutants the respective values of pKa of the proton release group in the unphotolyzed pigment and in M are approximately 8 and 7.5 in R82Q (in 1 M salt) and approximately 8 and 6.5 in R82K (in 150 mM KCl). Thus in R82Q the pKa of the proton release group does not decrease enough in the photocycle to allow early proton release from this group at neutral pH. 3) Early proton release in R82Q can be detected as a photocurrent signal that is kinetically distinct from those photocurrents that are due to proton movements from the Schiff base to D85 during M formation and from D96 to the Schiff base during the M-->N transition. 4) In R82Q, at neutral pH, proton uptake from the medium occurs during the formation of O. The proton is released during the O-->bacteriorhodopsin transition, probably from D85 because the normal proton release group cannot deprotonate at this pH. 5) The time constant of early proton release is increased from 85

  20. Fatty acids in Chinese edible oils: value of direct analysis as a basis for labeling.

    PubMed

    Wallingford, John C; Yuhas, Rebecca; Du, Shufa; Zhai, Fengying; Popkin, Barry M

    2004-12-01

    Edible oil is an important element in the diet of most transitional countries; nevertheless, little is known about the fatty acid composition of these oils. We examined the consumption of edible oils and the fatty acid composition of these oils obtained from a market survey conducted in seven Chinese provinces and in Beijing. Three days of measured household food intake from the 1997 China Health and Nutrition Survey households provided data on the consumption of edible oils. Edible oils sold in the capital cities of eight provinces were purchased. One hundred twenty-six samples, representing 14 different oils according to their labels, were assayed for their fatty acid content in 2001. Fatty acids were analyzed by standard gas chromatographic methods. More than 76% of households in China consume edible oil, providing an average of 29.6 g of edible oil per day to persons aged two years or older. Rapeseed was consumed by one-quarter of individuals. Rapeseed is rich in C22:1n9 cis (erucic acid). About 33% of edible oils differed from their labeled identification. Rapeseed oil, identified by the presence of C22:1n9 (erucic acid), was most frequently not labeled as such. In another 28% of the samples, trans isomers of linolenic acid were detected. Deviations from the label identification were more common in southern than in northern provinces. Regulations requiring complete labeling of mixed edible oils in China might help prevent unintentional consumption of fatty acids associated with adverse health outcomes. In particular, consumption of erucic acid and trans fatty acids might be reduced. The results suggest the need for closer control of food oil labeling in China, especially in the South. PMID:15646310

  1. Lowering the pKa of a bisimidazoline lead with halogen atoms results in improved activity and selectivity against Trypanosoma brucei in vitro.

    PubMed

    Ríos Martínez, Carlos H; Nué Martínez, J Jonathan; Ebiloma, Godwin U; de Koning, Harry P; Alkorta, Ibon; Dardonville, Christophe

    2015-08-28

    Diphenyl-based bis(2-iminoimidazolidines) are promising antiprotozoal agents that are curative in mouse models of stage 1 trypanosomiasis but devoid of activity in the late-stage disease, possibly due to poor brain penetration caused by their dicationic nature. We present here a strategy consisting in reducing the pKa of the basic 2-iminoimidazolidine groups though the introduction of chlorophenyl, fluorophenyl and pyridyl ring in the structure of the trypanocidal lead 4-(imidazolidin-2-ylideneamino)-N-(4-(imidazolidin-2-ylideneamino)phenyl)benzamide (1). The new compounds showed reduced pKa values (in the range 1-3 pKa units) for the imidazolidine group linked to the substituted phenyl ring. In vitro activities (EC50) against wild type and resistant strains of T. b. brucei (s427 and B48, respectively) were in the submicromolar range with four compounds being more active and selective than 1 (SI > 340). In particular, the two most potent compounds (3b and 5a) acted approximately 6-times faster than 1 to kill trypanosomes in vitro. No cross-resistance with the diamidine and melaminophenyl class of trypanocides was observed indicating that these compounds represent interesting leads for further in vivo studies.

  2. Universal quantitative kinase assay based on diagonal SCX chromatography and stable isotope dimethyl labeling provides high-definition kinase consensus motifs for PKA and human Mps1.

    PubMed

    Hennrich, Marco L; Marino, Fabio; Groenewold, Vincent; Kops, Geert J P L; Mohammed, Shabaz; Heck, Albert J R

    2013-05-01

    In order to understand cellular signaling, a clear understanding of kinase-substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates in vitro and in vivo. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome.

  3. Selected nutrient contents, fatty acid composition, including conjugated linoleic acid, and retention values in separable lean from lamb rib loins as affected by external fat and cooking method.

    PubMed

    Badiani, Anna; Montellato, Lara; Bochicchio, Davide; Anfossi, Paola; Zanardi, Emanuela; Maranesi, Magda

    2004-08-11

    Proximate composition and fatty acid profile, conjugated linoleic acid (CLA) isomers included, were determined in separable lean of raw and cooked lamb rib loins. The cooking methods compared, which were also investigated for cooking yields and true nutrient retention values, were dry heating of fat-on cuts and moist heating of fat-off cuts; the latter method was tested as a sort of dietetic approach against the more traditional former type. With significantly (P < 0.05) lower cooking losses, dry heating of fat-on rib-loins produced slightly (although only rarely significantly) higher retention values for all of the nutrients considered, including CLA isomers. On the basis of the retention values obtained, both techniques led to a minimum migration of lipids into the separable lean, which was higher (P < 0.05) in dry heating than in moist heating, and was characterized by the prevalence of saturated and monounsaturated fatty acids. On the whole, the response to cooking of the class of CLA isomers (including that of the nutritionally most important isomer cis-9,trans-11) was more similar to that of the monounsaturated than the polyunsaturated fatty acids.

  4. Acid-base properties of water-soluble organic matter of forest soils, studied by the pK-spectroscopy method.

    PubMed

    Shamrikova, E V; Ryazanov, M A; Vanchikova, E V

    2006-11-01

    Using the potentiometric titration and pK spectroscopy method, acid-base properties of water-soluble organic matter of forest soils have been studied. Five acidic classes composed of different substances with pK(a) values around 3.6; 4.8; 6.7; 8.7 and 9.7 have been identified. Testing the properties of soluble soil fraction, it is to be taken into account that when it is isolated from non-soluble soil matter, some water-soluble substances remain in soil and do not pass into the solution. Most firmly adsorbed in soil are water-soluble components with pK(a) 9.6-9.8.

  5. Genome-wide association and prediction of direct genomic breeding values for composition of fatty acids in Angus beef cattlea

    PubMed Central

    2013-01-01

    Background As consumers continue to request food products that have health advantages, it will be important for the livestock industry to supply a product that meet these demands. One such nutrient is fatty acids, which have been implicated as playing a role in cardiovascular disease. Therefore, the objective of this study was to determine the extent to which molecular markers could account for variation in fatty acid composition of skeletal muscle and identify genomic regions that harbor genetic variation. Results Subsets of markers on the Illumina 54K bovine SNPchip were able to account for up to 57% of the variance observed in fatty acid composition. In addition, these markers could be used to calculate a direct genomic breeding values (DGV) for a given fatty acids with an accuracy (measured as simple correlations between DGV and phenotype) ranging from -0.06 to 0.57. Furthermore, 57 1-Mb regions were identified that were associated with at least one fatty acid with a posterior probability of inclusion greater than 0.90. 1-Mb regions on BTA19, BTA26 and BTA29, which harbored fatty acid synthase, Sterol-CoA desaturase and thyroid hormone responsive candidate genes, respectively, explained a high percentage of genetic variance in more than one fatty acid. It was also observed that the correlation between DGV for different fatty acids at a given 1-Mb window ranged from almost 1 to -1. Conclusions Further investigations are needed to identify the causal variants harbored within the identified 1-Mb windows. For the first time, Angus breeders have a tool whereby they could select for altered fatty acid composition. Furthermore, these reported results could improve our understanding of the biology of fatty acid metabolism and deposition. PMID:24156620

  6. Investigation of the acid-base properties of mononitro-calix[4]arene with chemometric methods.

    PubMed

    Wang, Li; Shi, Xian-Fa; Zhu, Zhong-Liang

    2007-07-01

    The acid-base properties of mononitro-calix[4]arene was studied with chemometric methods by measurement of its UV absorbance under different pH. The chemometric method-iterative target transformation factor (ITTFA) was employed to resolve the acid-base fraction curves. Combining with other chemometric methods-principal component analysis (PCA) and evolving factor analysis (EFA), the proton dissociation behavior of the derivative was investigated in detail. The pK(a) values of the derivative were determined and the fraction curves and pure absorbing spectra of each absorbing component were obtained.

  7. Cell-type specific expression of a dominant negative PKA mutation in mice.

    PubMed

    Willis, Brandon S; Niswender, Colleen M; Su, Thomas; Amieux, Paul S; McKnight, G Stanley

    2011-01-01

    We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology. PMID:21533282

  8. PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease

    PubMed Central

    Bram, Zakariae; Louiset, Estelle; Renouf, Sylvie; Duparc, Céline; Boutelet, Isabelle; Rizk-Rabin, Marthe; Libé, Rossella; Young, Jacques; Carson, Dennis; Vantyghem, Marie-Christine; Szarek, Eva; Martinez, Antoine; Stratakis, Constantine A.; Bertherat, Jérôme

    2016-01-01

    Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD.

  9. PKA-Type I Selective Constrained Peptide Disruptors of AKAP Complexes

    PubMed Central

    Wang, Yuxiao; Ho, Tienhuei G.; Franz, Eugen; Hermann, Jennifer S.; Smith, F. Donelson; Hehnly, Heidi; Esseltine, Jessica L.; Hanold, Laura E.; Murph, Mandi M.; Bertinetti, Daniela; Scott, John D.; Herberg, Friedrich W.; Kennedy, Eileen J.

    2015-01-01

    A-Kinase Anchoring Proteins (AKAPs) coordinate complex signaling events by serving as spatiotemporal modulators of cAMP-dependent protein kinase activity in cells. Although AKAPs organize a plethora of diverse pathways, their cellular roles are often elusive due to the dynamic nature of these signaling complexes. AKAPs can interact with the type I or type II PKA holoenzymes by virtue of high-affinity interactions with the R-subunits. As a means to delineate AKAP-mediated PKA signaling in cells, we sought to develop isoform-selective disruptors of AKAP signaling. Here, we report the development of conformationally constrained peptides named RI-STapled Anchoring Disruptors (RI-STADs) that target the docking/dimerization domain of the type 1 regulatory subunit of PKA. These high-affinity peptides are isoform-selective for the RI isoforms, can outcompete binding by the classical AKAP disruptor Ht31, and can selectively displace RIα, but not RIIα, from binding the dual-specific AKAP149 complex. Importantly, these peptides are cell-permeable and disrupt Type I PKA-mediated phosphorylation events in the context of live cells. Hence, RI-STAD peptides are versatile cellular tools to selectively probe anchored type I PKA signaling events. PMID:25765284

  10. PKA regulatory subunit 1A inactivating mutation induces serotonin signaling in primary pigmented nodular adrenal disease

    PubMed Central

    Bram, Zakariae; Louiset, Estelle; Renouf, Sylvie; Duparc, Céline; Boutelet, Isabelle; Rizk-Rabin, Marthe; Libé, Rossella; Young, Jacques; Carson, Dennis; Vantyghem, Marie-Christine; Szarek, Eva; Martinez, Antoine; Stratakis, Constantine A.; Bertherat, Jérôme

    2016-01-01

    Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene, which induces constitutive activation of PKA in adrenocortical cells. Hypercortisolism is thought to result from PKA hyperactivity, but PPNAD tissues exhibit features of neuroendocrine differentiation, which may lead to stimulation of steroidogenesis by abnormally expressed neurotransmitters. We hypothesized that serotonin (5-HT) may participate in the pathophysiology of PPNAD-associated hypercortisolism. We show that PPNAD tissues overexpress the 5-HT synthesizing enzyme tryptophan hydroxylase type 2 (Tph2) and the serotonin receptors types 4, 6, and 7, leading to formation of an illicit stimulatory serotonergic loop whose pharmacological inhibition in vitro decreases cortisol production. In the human PPNAD cell line CAR47, the PKA inhibitor H-89 decreases 5-HT4 and 5-HT7 receptor expression. Moreover, in the human adrenocortical cell line H295R, inhibition of PRKAR1A expression increases the expression of Tph2 and 5-HT4/6/7 receptors, an effect that is blocked by H-89. These findings show that the serotonergic process observed in PPNAD tissues results from PKA activation by PRKAR1A mutations. They also suggest that Tph inhibitors may represent efficient treatments of hypercortisolism in patients with PPNAD. PMID:27699247

  11. Fast high-throughput method for the determination of acidity constants by capillary electrophoresis: I. Monoprotic weak acids and bases.

    PubMed

    Fuguet, Elisabet; Ràfols, Clara; Bosch, Elisabeth; Rosés, Martí

    2009-04-24

    A new and fast method to determine acidity constants of monoprotic weak acids and bases by capillary zone electrophoresis based on the use of an internal standard (compound of similar nature and acidity constant as the analyte) has been developed. This method requires only two electrophoretic runs for the determination of an acidity constant: a first one at a pH where both analyte and internal standard are totally ionized, and a second one at another pH where both are partially ionized. Furthermore, the method is not pH dependent, so an accurate measure of the pH of the buffer solutions is not needed. The acidity constants of several phenols and amines have been measured using internal standards of known pK(a), obtaining a mean deviation of 0.05 pH units compared to the literature values. PMID:19168179

  12. Fast high-throughput method for the determination of acidity constants by capillary electrophoresis: I. Monoprotic weak acids and bases.

    PubMed

    Fuguet, Elisabet; Ràfols, Clara; Bosch, Elisabeth; Rosés, Martí

    2009-04-24

    A new and fast method to determine acidity constants of monoprotic weak acids and bases by capillary zone electrophoresis based on the use of an internal standard (compound of similar nature and acidity constant as the analyte) has been developed. This method requires only two electrophoretic runs for the determination of an acidity constant: a first one at a pH where both analyte and internal standard are totally ionized, and a second one at another pH where both are partially ionized. Furthermore, the method is not pH dependent, so an accurate measure of the pH of the buffer solutions is not needed. The acidity constants of several phenols and amines have been measured using internal standards of known pK(a), obtaining a mean deviation of 0.05 pH units compared to the literature values.

  13. Tables of critical values for examining compositional non-randomness in proteins and nucleic acids

    NASA Technical Reports Server (NTRS)

    Laird, M.; Holmquist, R.

    1975-01-01

    A binomially distributed statistic is defined to show whether or not the proportion of a particular amino acid in a protein deviates from random expectation. An analogous statistic is derived for nucleotides in nucleic acids. These new statistics are simply related to the classical chi-squared test. They explicitly account for the compositional fluctuations imposed by the finite length of proteins, and they are more accurate than previous tables.

  14. Interpretation of pH-activity profiles for acid-base catalysis from molecular simulations.

    PubMed

    Dissanayake, Thakshila; Swails, Jason M; Harris, Michael E; Roitberg, Adrian E; York, Darrin M

    2015-02-17

    The measurement of reaction rate as a function of pH provides essential information about mechanism. These rates are sensitive to the pK(a) values of amino acids directly involved in catalysis that are often shifted by the enzyme active site environment. Experimentally observed pH-rate profiles are usually interpreted using simple kinetic models that allow estimation of "apparent pK(a)" values of presumed general acid and base catalysts. One of the underlying assumptions in these models is that the protonation states are uncorrelated. In this work, we introduce the use of constant pH molecular dynamics simulations in explicit solvent (CpHMD) with replica exchange in the pH-dimension (pH-REMD) as a tool to aid in the interpretation of pH-activity data of enzymes and to test the validity of different kinetic models. We apply the methods to RNase A, a prototype acid-base catalyst, to predict the macroscopic and microscopic pK(a) values, as well as the shape of the pH-rate profile. Results for apo and cCMP-bound RNase A agree well with available experimental data and suggest that deprotonation of the general acid and protonation of the general base are not strongly coupled in transphosphorylation and hydrolysis steps. Stronger coupling, however, is predicted for the Lys41 and His119 protonation states in apo RNase A, leading to the requirement for a microscopic kinetic model. This type of analysis may be important for other catalytic systems where the active forms of the implicated general acid and base are oppositely charged and more highly correlated. These results suggest a new way for CpHMD/pH-REMD simulations to bridge the gap with experiments to provide a molecular-level interpretation of pH-activity data in studies of enzyme mechanisms.

  15. Phenothiazines solution complexity - Determination of pKa and solubility-pH profiles exhibiting sub-micellar aggregation at 25 and 37°C.

    PubMed

    Pobudkowska, Aneta; Ràfols, Clara; Subirats, Xavier; Bosch, Elisabeth; Avdeef, Alex

    2016-10-10

    The ionization constants (pKa) and the pH-dependent solubility (log S-pH) of six phenothiazine derivatives (promazine hydrochloride, chlorpromazine hydrochloride, triflupromazine hydrochloride, fluphenazine dihydrochloride, perphenazine free base, and trifluoperazine dihydrochloride) were determined at 25 and 37°C. The pKa values of these low-soluble surface active molecules were determined by the cosolvent method (n-propanol/water at 37°C and methanol/water at 25°C). The log S-pH profiles were measured at 24h incubation time in 0.15M phosphate buffers. The log S-pH "shape-template" method, which critically depends on accurate pKa values (determined independently of solubility data), was used to propose speciation models, which were subsequently refined by rigorous mass-action weighted regression procedure described recently. Differential scanning calorimetry (DSC), UV-visible spectrophotometry, potentiometric, and high performance liquid chromatography (HPLC) measurements were used to characterize the compounds. The intrinsic solubility (S0) values of the three least-soluble drugs (chlorpromazine·HCl, triflupromazine·HCl, and trifluoperazine·2HCl) at 25°C were 0.5, 1.1, and 2.7μg/mL (resp.). These values increased to 5.5, 9.2, and 8.7μg/mL (resp.) at the physiological temperature. The enthalpies of solution for the latter compounds were exceptionally high positive (endothermic) values (99-152kJ·mol(-1)). Cationic sub-micellar aggregates were evident (from the distortions in the log S-pH profiles) for chlorpromazine, fluphenazine, perphenazine, and trifluoperazine at 25°C. The effects persisted at 37°C for chlorpromazine and trifluoperazine. The solids in suspension were apparently amorphous in cases where the drugs were introduced as the chloride salts.

  16. Involvement of PKA and HO-1 signaling in anti-inflammatory effects of surfactin in BV-2 microglial cells

    SciTech Connect

    Park, Sun Young; Kim, Ji-Hee; Lee, Sang Joon; Kim, YoungHee

    2013-04-01

    Surfactin, one of the most powerful biosurfactants, is a bacterial cyclic lipopeptide. Here, we investigated the anti-neuroinflammatory properties of surfactin in lipoteichoic acid (LTA)-stimulated BV-2 microglial cells. Surfactin significantly inhibited excessive production of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), prostaglandin E{sub 2} (PGE{sub 2}), nitric oxide (NO) and reactive oxygen species (ROS), and suppressed the expression of matrix metalloproteinase-9 (MMP-9), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent mechanistic studies revealed that surfactin inhibited LTA-induced nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription-1 (STAT-1) activation. However, surfactin increases the phosphorylation of the STAT-3, a component of the homeostatic mechanism causing anti-inflammatory events. We also demonstrated that surfactin induces heme oxygenase-1 (HO-1) expression and nuclear factor-regulated factor-2 (Nrf-2) activation, and that the anti-inflammatory effects of surfactin are abrogated by small interfering RNA-mediated knock-down of HO-1 or Nrf-2. Interestingly, we found that surfactin increased the level of cAMP and induced phosphorylation of cAMP responsive element binding protein (CREB) in microglial cells. Furthermore, treatment with the protein kinase A (PKA) inhibitor, H-89, blocked HO-1 induction by surfactin and abolished surfactin's suppressive effects on ROS and NO production. These results indicate that HO-1 and its upstream effector, PKA, play a pivotal role in the anti-neuroinflammatory response of surfactin in LTA-stimulated microglia. Therefore, surfactin might have therapeutic potential for neuroprotective agents to treat inflammatory and neurodegenerative diseases. - Highlights: ► Surfactin inhibits proinflammatory mediator synthesis in LTA-activated BV-2 cells. ► Surfactin suppresses NF-κB and STAT-1, but potentiates

  17. Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype

    PubMed Central

    Gilbert, Merle L.; Yang, Linghai; Su, Thomas

    2015-01-01

    PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus. PMID:25587115

  18. Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype.

    PubMed

    Gilbert, Merle L; Yang, Linghai; Su, Thomas; McKnight, G Stanley

    2015-03-15

    PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 (Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.

  19. Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα.

    PubMed

    Akimoto, Madoka; McNicholl, Eric Tyler; Ramkissoon, Avinash; Moleschi, Kody; Taylor, Susan S; Melacini, Giuseppe

    2015-01-01

    Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a "gatekeeper" domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91-379) in its apo, cAMP2, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics.

  20. Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα

    PubMed Central

    Akimoto, Madoka; McNicholl, Eric Tyler; Ramkissoon, Avinash; Moleschi, Kody; Taylor, Susan S.; Melacini, Giuseppe

    2015-01-01

    Protein Kinase A (PKA) is the major receptor for the cyclic adenosine monophosphate (cAMP) secondary messenger in eukaryotes. cAMP binds to two tandem cAMP-binding domains (CBD-A and -B) within the regulatory subunit of PKA (R), unleashing the activity of the catalytic subunit (C). While CBD-A in RIα is required for PKA inhibition and activation, CBD-B functions as a “gatekeeper” domain that modulates the control exerted by CBD-A. Preliminary evidence suggests that CBD-B dynamics are critical for its gatekeeper function. To test this hypothesis, here we investigate by Nuclear Magnetic Resonance (NMR) the two-domain construct RIα (91–379) in its apo, cAMP2, and C-bound forms. Our comparative NMR analyses lead to a double conformational selection model in which each apo CBD dynamically samples both active and inactive states independently of the adjacent CBD within a nearly degenerate free energy landscape. Such degeneracy is critical to explain the sensitivity of CBD-B to weak interactions with C and its high affinity for cAMP. Binding of cAMP eliminates this degeneracy, as it selectively stabilizes the active conformation within each CBD and inter-CBD contacts, which require both cAMP and W260. The latter is contributed by CBD-B and mediates capping of the cAMP bound to CBD-A. The inter-CBD interface is dispensable for intra-CBD conformational selection, but is indispensable for full activation of PKA as it occludes C-subunit recognition sites within CBD-A. In addition, the two structurally homologous cAMP-bound CBDs exhibit marked differences in their residual dynamics profiles, supporting the notion that conservation of structure does not necessarily imply conservation of dynamics. PMID:26618408

  1. The Ka Values of Water and the Hydronium Ion for Comparison with Other Acids.

    ERIC Educational Resources Information Center

    Campbell, Mark L.; Waite, Boyd A.

    1990-01-01

    Provided is a rebuttal to an argument concerning the use of Ka values for water and the hydronium ion. The derivation of new values as a result of the treatment of the water as the solvent and using a Raoult's Law standard state is discussed. (CW)

  2. Enhancing essential amino acids and health benefit components in grain crops for improved nutritional values.

    PubMed

    Wenefrida, Ida; Utomo, Herry S; Blanche, Sterling B; Linscombe, S D

    2009-01-01

    Improving essential amino acids or protein content, along with other phytonutrients in the food crops, will affect a great portion of the world population, especially in developing countries where rice grain is the main source of protein. Malnutrition, including deficiencies in protein/energy, iron/zinc, vitamin A, and iodine, causes a total 24,000 deaths per day worldwide. The problem is severe where rice is the major staple food. Protein deficiency involves both the quantity (amount) and quality (the content in essential amino acids) of the dietary protein. Various interventions, such as distribution, fortification, dietary diversification, and measures against infectious diseases, have been applied to reduce deficiency disorders. The problem, however, remains unsolved. Developing genetically novel lines with elevated content of essential amino acids together with other health benefit components becomes more feasible for the enhancement of breeding techniques, genomics, molecular manipulations, and genetic engineering. Advancement in basic genetic and genetic engineering has resulted in successful enrichment of some essential amino acids, such as lysine (Lys), tryptophan (Trp), and methionine (Met). Successful genetic enhancement has been largely restricted to the maize crop through enrichment of grain Lys and to some extends Trp. Since rice is the main source of calories and protein intake for billions of people, enhancing essential amino acids in rice represents a tremendous challenge. This paper will discuss and review the current status in basic genetics, molecular genetics, and genetic engineering associated with the enhancement of amino acids and other health benefit components in major grain crop improvement. Patents and future efforts associated with enhancing nutritional quality of the grain will also be reviewed as a concerted effort to solve the malnutrition problem and improve the quality of life worldwide.

  3. Value-added lipid production from brown seaweed biomass by two-stage fermentation using acetic acid bacterium and thraustochytrid.

    PubMed

    Arafiles, Kim Hazel V; Iwasaka, Hiroaki; Eramoto, Yuri; Okamura, Yoshiko; Tajima, Takahisa; Matsumura, Yukihiko; Nakashimada, Yutaka; Aki, Tsunehiro

    2014-11-01

    Thraustochytrid production of polyunsaturated fatty acids and xanthophylls have been generally sourced from crop-derived substrates, making the exploration of alternative feedstocks attractive since they promise increased sustainability and lower production costs. In this study, a distinct two-stage fermentation system was conceptualized for the first time, using the brown seaweed sugar mannitol as substrate for the intermediary biocatalyst Gluconobacter oxydans, an acetic acid bacterium, along with the marine thraustochytrid Aurantiochytrium sp. to produce the value-added lipids and xanthophylls. Jar fermenter culture resulted in seaweed mannitol conversion to fructose with an efficiency of 83 % by G. oxydans and, after bacteriostasis with sea salts, production of astaxanthin and docosahexaenoic acid by Aurantiochytrium sp. KH105. Astaxanthin productivity was high at 3.60 mg/L/day. This new system, therefore, widens possibilities of obtaining more varieties of industrially valuable products including foods, cosmetics, pharmaceuticals, and biofuel precursor lipids from seaweed fermentation upon the use of suitable thraustochytrid strains.

  4. VALUING ACID MINE DRAINAGE REMEDIATION IN WEST VIRGINIA: A HEDONIC MODELING APPROACH

    EPA Science Inventory

    States with active and abandoned mines face large private and public costs to remediate damage to streams and rivers from acid mine drainage (AMD). Appalachian states have an especially large number of contaminated streams and rivers, and the USGS places AMD as the primary source...

  5. The economic value of pelargonic acid as a natural herbicide in sweet bell peppers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Economic return on investment in respect to weed control management practices continue to be an essential element in use of naturally occurring substances for weed control in vegetable production. Pelargonic acid, although not certified as organic, is naturally occurring in many plants, animals, and...

  6. VALUING ACID MINE DRAINAGE REMEDIATION IN WEST VIRGINIA: BENEFIT TRANSFER WITH PREFERENCE CALIBRATION

    EPA Science Inventory

    Several thousand kilometers of West Virginia streams are degraded by acid mine drainage (AMD), and the estimates for cleanup range in the billions of dollars. Not enough money is available to restore all the affected streams, so some way to prioritize those streams is needed. Ben...

  7. VALUING ACID MINE DRAINAGE REMEDIATION OF IMPAIRED WATERWAYS IN WEST VIRGINIA: A HEDONIC MODELING APPROACH

    EPA Science Inventory

    States with active and abandoned mines face large private and public costs to remediate damage to streams and rivers from acid mine drainage (AMD), the metal rich runoff flowing primarily from abandoned mines and surface deposits of mine waste. AMD can lower stream and river pH ...

  8. VALUING ACID MINE DRAINAGE REMEDIATION IN WEST VIRGINIA: A HEDONIC MODELING APPROACH INCORPORATING GEOGRAPHIC INFORMATION SYSTEMS

    EPA Science Inventory

    States with active and abandoned mines face large private and public costs to remediate damage to streams and rivers from acid mine drainage (AMD). Appalachian states have an especially large number of contaminated streams and rivers, and the USGS places AMD as the primary source...

  9. Relativistic effects on acidities and basicities of Brønsted acids and bases containing gold.

    PubMed

    Koppel, Ilmar A; Burk, Peeter; Kasemets, Kalev; Koppel, Ivar

    2013-11-01

    It is usually believed that relativistic effects as described by the Dirac-Schrödinger equation (relative to the classical or time-independent Schrödinger equation) are of little importance in chemistry. A closer look, however, reveals that some important and widely known properties (e.g., gold is yellow, mercury is liquid at room temperature) stem from relativistic effects. So far the influence of relativistic effects on the acid-base properties has been mostly ignored. Here we show that at least for compounds of gold such omission is completely erroneous and would lead to too high basicity and too low acidity values with errors in the range of 25-55 kcal mol(-1) (or 20 to 44 powers of ten in pK(a) units) in the gas-phase. These findings have important implications for the design of new superstrong acids and bases, and for the understanding of gold-catalysed reactions.

  10. Diet-related reference values for plasma amino acids in newborns measured by reversed-phase HPLC.

    PubMed

    Scott, P H; Sandham, S; Balmer, S E; Wharton, B A

    1990-11-01

    We have measured by reversed-phase HPLC concentrations of amino acids in plasma in groups of 80 normal appropriate-weight term babies fed from birth either a casein formula (WhiteCap SMA, n = 26), a whey formula (Gold Cap SMA, n = 26), or breast milk (n = 28). They were studied from day 11 to week 15 postpartum. The trend was towards an increase in amino acid concentrations in plasma with age, more marked in formula-fed than in breast-fed infants. Reference values were derived for each group. Both formula-fed groups showed several differences from the breast-fed group. Detailed examination indicated that tyrosine, phenylalanine, and methionine concentrations were increased in the casein-fed group greater than 20% of the time, but only threonine was similarly increased in the whey-fed group. Other amino acids, different ones for each formula group, were increased less frequently. There were no consistent correlations with any aspect of infant growth. Appropriate reference values are important for interpreting amino acid concentrations in plasma from newborns and for evaluating the effects of any future dietary modifications to infant formulas. HPLC analysis provides a suitable highly sensitive method for undertaking such studies.

  11. Acid-base strength and acidochromism of some dimethylamino-azinium iodides. An integrated experimental and theoretical study.

    PubMed

    Benassi, Enrico; Carlotti, Benedetta; Fortuna, Cosimo G; Barone, Vincenzo; Elisei, Fausto; Spalletti, Anna

    2015-01-15

    The effects of pH on the spectral properties of stilbazolium salts bearing dimethylamino substituents, namely, trans isomers of the iodides of the dipolar E-[2-(4-dimethylamino)styryl]-1-methylpyridinium, its branched quadrupolar analogue E,E-[2,6-di-(p-dimethylamino)styryl]-1-methylpyridinium, and three analogues, chosen to investigate the effects of the stronger quinolinium acceptor, the longer butadiene π bridge, or both, were investigated through a joint experimental and computational approach. A noticeable acidochromism of the absorption spectra (interesting for applications) was observed, with the basic and protonated species giving intensely colored and transparent solutions, respectively. The acid–base equilibrium constants for the protonation of the dimethylamino group in the ground state (pKa) were experimentally derived. Theoretical calculations according to the thermodynamic Born-Haber cycle provided pKa values in good agreement with the experimental values. The very low fluorescence yield did not allow a direct investigation of the changes in the acid-base properties in the excited state (pKa*) by fluorimetric titrations. Their values were derived by quantum-mechanical calculations and estimated experimentally on the basis of the Förster cycle.

  12. Aqueous infrared carboxylate absorbances: Aliphatic di-acids

    USGS Publications Warehouse

    Cabaniss, S.E.; Leenheer, J.A.; McVey, I.F.

    1998-01-01

    Aqueous attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of 18 aliphatic di-carboxylic acids are reported as a function of pH. The spectra show isosbestic points and intensity changes which indicate that Beer's law is obeyed, and peak frequencies lie within previously reported ranges for aqueous carboxylates and pure carboxylic acids. Intensity sharing from the symmetric carboxylate stretch is evident in many cases, so that bands which are nominally due to alkyl groups show increased intensity at higher pH. The asymmetric stretch of the HA- species is linearly related to the microscopic acidity constant of the H2A species, with ??pK 2 intervening atoms). The results suggest that aqueous ATR-FTIR may be able to estimate 'intrinsic' pKa values of carboxylic acids, in addition to providing quantitative estimates of ionization. ?? 1998 Elsevier Science B.V. All rights reserved.

  13. Solvent effects in acid-catalyzed biomass conversion reactions.

    PubMed

    Mellmer, Max A; Sener, Canan; Gallo, Jean Marcel R; Luterbacher, Jeremy S; Alonso, David Martin; Dumesic, James A

    2014-10-27

    Reaction kinetics were studied to quantify the effects of polar aprotic organic solvents on the acid-catalyzed conversion of xylose into furfural. A solvent of particular importance is γ-valerolactone (GVL), which leads to significant increases in reaction rates compared to water in addition to increased product selectivity. GVL has similar effects on the kinetics for the dehydration of 1,2-propanediol to propanal and for the hydrolysis of cellobiose to glucose. Based on results obtained for homogeneous Brønsted acid catalysts that span a range of pKa values, we suggest that an aprotic organic solvent affects the reaction kinetics by changing the stabilization of the acidic proton relative to the protonated transition state. This same behavior is displayed by strong solid Brønsted acid catalysts, such as H-mordenite and H-beta. PMID:25214063

  14. One-step production of biodiesel from Jatropha oil with high-acid value in ionic liquids.

    PubMed

    Guo, Feng; Fang, Zhen; Tian, Xiao-Fei; Long, Yun-Duo; Jiang, Li-Qun

    2011-06-01

    Catalytic conversion of un-pretreated Jatropha oil with high-acid value (13.8 mg KOH/g) to biodiesel was studied in ionic liquids (ILs) with metal chlorides. Several commercial ILs were used to catalyze the esterification of oleic acid. It was found that 1-butyl-3-methylimidazolium tosylate ([BMIm][CH(3)SO(3)]; a Brønsted acidic IL) had the highest catalytic activity with 93% esterification rate for oleic acid at 140°C but only 12% biodiesel yield at 120°C. When FeCl(3) was added to [BMIm][CH(3)SO(3)], a maximum biodiesel yield of 99.7% was achieved at 120°C. Because metal ions in ILs supplied Lewis acidic sites, and more of the sites could be provided by trivalent metallic ions than those of bivalent ones. It was also found that the catalytic activity with bivalent metallic ions increased with atomic radius. Mixture of [BMIm][CH(3)SO(3)] and FeCl(3) was easily separated from products for reuse to avoid producing pollutants. PMID:21420854

  15. Fluoro- and perfluoralkylsulfonylpentafluoroanilides: synthesis and characterization of NH acids for weakly coordinating anions and their gas-phase and solution acidities.

    PubMed

    Kögel, Julius F; Linder, Thomas; Schröder, Fabian G; Sundermeyer, Jörg; Goll, Sascha K; Himmel, Daniel; Krossing, Ingo; Kütt, Karl; Saame, Jaan; Leito, Ivo

    2015-04-01

    Fluoro- and perfluoralkylsulfonyl pentafluoroanilides [HN(C6F5)(SO2X); X = F, CF3, C4F9, C8F17] are a class of imides with two different strongly electron-withdrawing substituents attached to a nitrogen atom. They are NH acids, the unsymmetrical hybrids of the well-known symmetrical bissulfonylimides and bispentafluorophenylamine. The syntheses, the structures of these perfluoroanilides, their solvates, and some selected lithium salts give rise to a structural variety beyond the symmetrical parent compounds. The acidities of representative subsets of these novel NH acids have been investigated experimentally and quantum-chemically and their gas-phase acidities (GAs) are reported, as well as the pKa values of these compounds in acetonitrile (MeCN) and DMSO solution. In quantum chemical investigations with the vertical and relaxed COSMO cluster-continuum models (vCCC/rCCC), the unusual situation is encountered that the DMSO-solvated acid Me2SO-H-N(SO2CF3)2, optimized in the gas phase (vCCC model), dissociates to Me2SO-H(+)-N(SO2CF3)2(-) during structural relaxation and full optimization with the solvation model turned on (rCCC model). This proton transfer underlines the extremely high acidity of HN(SO2CF3)2. The importance of this effect is studied computationally in DMSO and MeCN solution. Usually this effect is less pronounced in MeCN and is of higher importance in the more basic solvent DMSO. Nevertheless, the neglect of the structural relaxation upon solvation causes typical changes in the computational pKa values of 1 to 4 orders of magnitude (4-20 kJ mol(-1)). The results provide evidence that the published experimental DMSO pKa value of HN(SO2CF3)2 should rather be interpreted as the pKa of a Me2SO-H(+)-N(SO2CF3)2(-) contact ion pair. PMID:25727401

  16. Fluoro- and perfluoralkylsulfonylpentafluoroanilides: synthesis and characterization of NH acids for weakly coordinating anions and their gas-phase and solution acidities.

    PubMed

    Kögel, Julius F; Linder, Thomas; Schröder, Fabian G; Sundermeyer, Jörg; Goll, Sascha K; Himmel, Daniel; Krossing, Ingo; Kütt, Karl; Saame, Jaan; Leito, Ivo

    2015-04-01

    Fluoro- and perfluoralkylsulfonyl pentafluoroanilides [HN(C6F5)(SO2X); X = F, CF3, C4F9, C8F17] are a class of imides with two different strongly electron-withdrawing substituents attached to a nitrogen atom. They are NH acids, the unsymmetrical hybrids of the well-known symmetrical bissulfonylimides and bispentafluorophenylamine. The syntheses, the structures of these perfluoroanilides, their solvates, and some selected lithium salts give rise to a structural variety beyond the symmetrical parent compounds. The acidities of representative subsets of these novel NH acids have been investigated experimentally and quantum-chemically and their gas-phase acidities (GAs) are reported, as well as the pKa values of these compounds in acetonitrile (MeCN) and DMSO solution. In quantum chemical investigations with the vertical and relaxed COSMO cluster-continuum models (vCCC/rCCC), the unusual situation is encountered that the DMSO-solvated acid Me2SO-H-N(SO2CF3)2, optimized in the gas phase (vCCC model), dissociates to Me2SO-H(+)-N(SO2CF3)2(-) during structural relaxation and full optimization with the solvation model turned on (rCCC model). This proton transfer underlines the extremely high acidity of HN(SO2CF3)2. The importance of this effect is studied computationally in DMSO and MeCN solution. Usually this effect is less pronounced in MeCN and is of higher importance in the more basic solvent DMSO. Nevertheless, the neglect of the structural relaxation upon solvation causes typical changes in the computational pKa values of 1 to 4 orders of magnitude (4-20 kJ mol(-1)). The results provide evidence that the published experimental DMSO pKa value of HN(SO2CF3)2 should rather be interpreted as the pKa of a Me2SO-H(+)-N(SO2CF3)2(-) contact ion pair.

  17. Conversion of Surplus Picric Acid/Explosive D to Higher Value Products

    SciTech Connect

    Mitchell, A R; Hsu, P C; Coburn, M D; Schmidt, R D; Pagoria, P F; Lee, G S; Kwak, S S W

    2003-02-28

    The global demilitarization of nuclear and conventional munitions is producing millions of pounds of surplus energetic materials. Historically, energetic materials (high explosives, propellants, and pyrotechnics) have been disposed of by open burning/open detonation (OB/OD). The use of OB/OD is becoming unacceptable due to public concerns and increasingly stringent environmental regulations. Clearly, there is a great need to develop environmentally sound and cost-effective alternatives to OB/OD. The conversion of surplus picric acid and/or ammonium picrate (Explosive D) to 1,3,5-triamino-2,4,6-trinitrobenzene (TATB) has been subject of extensive process development studies at Lawrence Livermore National Laboratory (LLNL). LLNL, under the direction and sponsorship of the U.S. Army Defense Ammunition Center (DAC), is developing a process for the conversion of picric acid to TATB on a pilot scale.

  18. Simple quantification of surface carboxylic acids on chemically oxidized multi-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Gong, Hyejin; Kim, Seong-Taek; Lee, Jong Doo; Yim, Sanggyu

    2013-02-01

    The surface of multi-walled carbon nanotube (MWCNT) was chemically oxidized using nitric acid and sulfuric-nitric acid mixtures. Thermogravimetric analysis, transmission electron microscopy and infrared spectroscopy revealed that the use of acid mixtures led to higher degree of oxidation. More quantitative identification of surface carboxylic acids was carried out using X-ray photoelectron spectroscopy (XPS) and acid-base titration. However, these techniques are costly and require very long analysis times to promptly respond to the extent of the reaction. We propose a much simpler method using pH measurements and pre-determined pKa value in order to estimate the concentration of carboxylic acids on the oxidized MWCNT surfaces. The results from this technique were consistent with those obtained from XPS and titration, and it is expected that this simple quantification method can provide a cheap and fast way to monitor and control the oxidation reaction of MWCNT.

  19. Effect of heme modification on oxygen affinity of myoglobin and equilibrium of the acid-alkaline transition in metmyoglobin.

    PubMed

    Shibata, Tomokazu; Nagao, Satoshi; Fukaya, Masashi; Tai, Hulin; Nagatomo, Shigenori; Morihashi, Kenji; Matsuo, Takashi; Hirota, Shun; Suzuki, Akihiro; Imai, Kiyohiro; Yamamoto, Yasuhiko

    2010-05-01

    Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyl (CF(3)) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O(2) affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF(3) substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by approximately 1 pH unit upon the introduction of one CF(3) group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O(2) affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P(50) value, which is the partial pressure of O(2) required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O(2) affinity upon the introduction of an electron-withdrawing CF(3) group is due to an increase in the O(2) dissociation rate. Since the introduction of a CF(3) group substitution is thought to prevent further Fe(2+)-O(2) bond polarization and hence formation of a putative Fe(3+)-O(2)(-)-like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O(2) dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O(2) binding to the protein, the CO

  20. PKA activation in concert with ARIS and asterosap induces the acrosome reaction in starfish.

    PubMed

    Islam, M Sadiqul; Kawase, O; Hase, S; Hoshi, M; Matsumoto, M

    2006-11-01

    The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.

  1. PKA Regulates PINK1 Stability and Parkin Recruitment to Damaged Mitochondria through Phosphorylation of MIC60.

    PubMed

    Akabane, Shiori; Uno, Midori; Tani, Naoki; Shimazaki, Shunta; Ebara, Natsumi; Kato, Hiroki; Kosako, Hidetaka; Oka, Toshihiko

    2016-05-01

    A mitochondrial kinase, PTEN-induced putative kinase 1 (PINK1), selectively recruits the ubiquitin ligase Parkin to damaged mitochondria, which modifies mitochondria by polyubiquitination, leading to mitochondrial autophagy. Here, we report that treatment with an adenylate cyclase agonist or expression of protein kinase A (PKA) impairs Parkin recruitment to damaged mitochondria and decreases PINK1 protein levels. We identified a mitochondrial membrane protein, MIC60 (also known as mitofilin), as a PKA substrate. Mutational and mass spectrometric analyses revealed that the Ser528 residue of MIC60 undergoes PKA-dependent phosphorylation. MIC60 transiently interacts with PINK1, and MIC60 downregulation leads to a reduction in PINK1 and mislocalization of Parkin. Phosphorylation-mimic mutants of MIC60 fail to restore the defect in Parkin recruitment in MIC60-knocked down cells, whereas a phosphorylation-deficient MIC60 mutant facilitates the mitochondrial localization of Parkin. Our findings indicate that PKA-mediated phosphorylation of MIC60 negatively regulates mitochondrial clearance that is initiated by PINK1 and Parkin. PMID:27153535

  2. Evolutionary paths of the cAMP-dependent protein kinase (PKA) catalytic subunits.

    PubMed

    Søberg, Kristoffer; Jahnsen, Tore; Rognes, Torbjørn; Skålhegg, Bjørn S; Laerdahl, Jon K

    2013-01-01

    3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods. PMID:23593352

  3. Ethanol Activation of PKA Mediates Single-Minded 2 Expression in Neuronal Cells.

    PubMed

    Wang, Xiaolan; Yang, Zhihua; Sun, Yinan; Zhou, Hanjing; Chu, Guangpin; Zhang, Jing; Meng, Xianfang

    2015-12-01

    Prenatal ethanol exposure can cause extensive apoptotic neurodegeneration throughout the developing central nervous system (CNS), which results in cognitive deficits and memory decline. However, the underlying mechanisms need further study. Single-minded 2 (Sim2), a transcriptional repressor, is reportedly involved in diseases that impair learning and memory, such as Down syndrome (DS) and Alzheimer's disease. It is still unknown whether Sim2 is involved in regulating ethanol-mediated neuronal injury that might ultimately lead to neuronal dysfunction and subsequent learning and memory deficits. To study the effects of ethanol on Sim2 expression and neuronal injury, we used animal models and cell culture experiments. Our results indicated that in SH-SY5Y cells, ethanol exposure increased Sim2 expression and levels of cleaved caspase 3, which is a marker for cells undergoing apoptosis. Silencing Sim2 expression attenuated caspase 3 activation and cellular apoptosis. We also found that protein kinase A (PKA) activation induced Sim2 expression, as did ethanol. Inhibiting the PKA signaling pathway with H-89 decreased Sim2 expression and cleavage of caspase 3 that was induced by ethanol in vivo and in vitro. We further found that PKA regulated Sim2 expression at the transcriptional level. These results demonstrate that ethanol leads to increased Sim2 expression via the PKA pathway, ultimately resulting in apoptotic cell death.

  4. Selectivity Mechanism of ATP-Competitive Inhibitors for PKB and PKA.

    PubMed

    Wu, Ke; Pang, Jingzhi; Song, Dong; Zhu, Ying; Wu, Congwen; Shao, Tianqu; Chen, Haifeng

    2015-07-01

    Protein kinase B (PKB) acts as a central node on the PI3K kinase pathway. Constitutive activation and overexpression of PKB have been identified to involve in various cancers. However, protein kinase A (PKA) sharing high homology with PKB is essential for metabolic regulation. Therefore, specific targeting on PKB is crucial strategy in drug design and development for antitumor. Here, we had revealed the selectivity mechanism for PKB inhibitors with molecular dynamics simulation and 3D-QSAR methods. Selective inhibitors of PKB could form more hydrogen bonds and hydrophobic contacts with PKB than those with PKA. This could explain that selective inhibitor M128 is more potent to PKB than to PKA. Then, 3D-QSAR models were constructed for these selective inhibitors and evaluated by test set compounds. 3D-QSAR model comparison of PKB inhibitors and PKA inhibitors reveals possible methods to improve the selectivity of inhibitors. These models can be used to design new chemical entities and make quantitative prediction of the specific selective inhibitors before resorting to in vitro and in vivo experiment.

  5. PKC and PKA inhibitors reinstate morphine-induced behaviors in morphine tolerant mice.

    PubMed

    Smith, Forrest L; Javed, Ruby R; Smith, Paul A; Dewey, William L; Gabra, Bichoy H

    2006-12-01

    Male Swiss Webster mice exhibited antinociception, hypothermia and Straub tail 3h following a 75mg morphine pellet implantation. These signs disappeared by 72h, and the morphine-pelleted mice were indistinguishable from placebo-pelleted ones, although brain morphine concentrations ranged from 200 to 400ng/gm. We previously demonstrated that chemical inhibitors of protein kinase C (PKC) and A (PKA) are able to reverse morphine tolerance in acutely morphine-challenged mice. However, it was not known whether the reversal of tolerance was due to the interaction of kinase inhibitors with the morphine released from the pellet, the acutely injected morphine to challenge tolerant mice, or both. The present study aimed at determining the interaction between the PKC and PKA inhibitors and the morphine released "solely" from the pellet to reinstate the morphine-induced behavioral and physiological effects, 72h after implantation of morphine pellets. Placebo or 75mg morphine pellets were surgically implanted, and testing was conducted 72h later. Our results showed that the intracerebroventricular (i.c.v.) administration of the PKC inhibitors, bisindolylmaleimide I and Gö-6976 as well as the PKA inhibitors, 4-cyano-3-methylisoquinoline and KT-5720, restored the morphine-induced behaviors of antinociception, Straub tail and hypothermia in morphine-pelleted mice to the same extent observed 3h following the pellet implantation. The tail withdrawal and the hot plate reaction time expressed as percent maximum possible effect (%MPE) was increased to 80-100 and 41-90%, respectively, in PKC and PKA inhibitor-treated morphine tolerant mice compared to 2-10% in non-treated mice. Similarly, a significant hypothermia (1.3-4.0 degrees C decrease in body temperature) was detected in PKC and PKA inhibitor-treated morphine tolerant mice compared to an euthermic state in non-treated morphine tolerant mice. Finally, the Straub tail score was increased to 1.1-1.6 in PKC and PKA inhibitor

  6. Cereal grain, rachis and pulse seed amino acid δ15N values as indicators of plant nitrogen metabolism.

    PubMed

    Styring, Amy K; Fraser, Rebecca A; Bogaard, Amy; Evershed, Richard P

    2014-01-01

    Natural abundance δ(15)N values of plant tissue amino acids (AAs) reflect the cycling of N into and within plants, providing an opportunity to better understand environmental and anthropogenic effects on plant metabolism. In this study, the AA δ(15)N values of barley (Hordeum vulgare) and bread wheat (Triticum aestivum) grains and rachis and broad bean (Vicia faba) and pea (Pisum sativum) seeds, grown at the experimental farm stations of Rothamsted, UK and Bad Lauchstädt, Germany, were determined by GC-C-IRMS. It was found that the δ(15)N values of cereal grain and rachis AAs could be largely attributed to metabolic pathways involved in their biosynthesis and catabolism. The relative (15)N-enrichment of phenylalanine can be attributed to its involvement in the phenylpropanoid pathway and glutamate has a δ(15)N value which is an average of the other AAs due to its central role in AA-N cycling. The relative AA δ(15)N values of broad bean and pea seeds were very different from one another, providing evidence for differences in the metabolic routing of AAs to the developing seeds in these leguminous plants. This study has shown that AA δ(15)N values relate to known AA biosynthetic pathways in plants and thus have the potential to aid understanding of how various external factors, such as source of assimilated N, influence metabolic cycling of N within plants.

  7. 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.

  8. LC50 values for rats acutely exposed to vapors of acrylic and methacrylic acid esters

    SciTech Connect

    Oberly, R.; Tansy, M.F.

    1985-01-01

    Acute exposure studies were conducted using adult male Sprague-Dawley rats to obtain LC50/24 concentrations for the common esters of acrylic and methacrylic acids. The order of acute toxicity was determined to be methyl acrylate > ethyl acrylate > butyl acrylate > butyl methacrylate > methyl methacrylate > ethyl methacrylate. Four-hour daily exposures (excluding weekends) of young adult male rats to 110 ppm methyl acrylate in air over a period of 32 d failed to produce significant differences in body or tissue weights, blood chemistries, gross metabolic performance, and spontaneous small-intestinal motor activities when compared with a sham-exposed group.

  9. REDUCTION OF PLUTONIUM VALUES IN AN ACIDIC AQUEOUS SOLUTION WITH FORMALDEHYDE

    DOEpatents

    Olson, C.M.

    1959-06-01

    A method is given for reducing Pu to the tetravalent state and lowering the high acidity of dissolver solutions containing U and Pu. Formaldehyde is added to the HNO/sub 3/ solution of U and Pu to effect a formaldehyde to HNO/sub 3/ molar ratio of 0.375:1 to 1.5:1. The Pu can then be removed from the solution by carrier precipitation using BiPO/sub 4/ or by ion exchange. (T.R.H.)

  10. Sex differences in behavioral and PKA cascade responses to repeated cocaine administration.

    PubMed

    Zhou, Luyi; Sun, Wei-Lun; Weierstall, Karen; Minerly, Ana Christina; Weiner, Jan; Jenab, Shirzad; Quinones-Jenab, Vanya

    2016-10-01

    Previous studies have shown sex different patterns in behavioral responses to cocaine. Here, we used between-subject experiment design to study whether sex differences exist in the development of behavioral sensitization and tolerance to repeated cocaine, as well as the role of protein kinase A (PKA) signaling cascade in this process. Ambulatory and rearing responses were recorded in male and female rats after 1 to 14 days of administration of saline or cocaine (15 mg/kg; ip). Correspondent PKA-associated signaling in the nucleus accumbens (NAc) and caudate-putamen (CPu) was measured at each time point. Our results showed that females exhibited higher cocaine-induced behavioral responses and developed behavioral sensitization and tolerance faster than males. Whereas females developed behavioral sensitization to cocaine after 2 days and tolerance after 14 days, male rats developed sensitization after 5 days. In addition, cocaine induced a sexual dimorphic pattern in the progression of neuronal adaptations on the PKA cascade signaling in region (NAc vs. CPu) and time (days of cocaine administration)-dependent manners. In general, more PKA signaling cascade changes were found in the NAc of males on day 5 and in the CPu of females with repeated cocaine injection. In addition, in females, behavioral activities positively correlated with FosB levels in the NAc and CPu and negatively correlated with Cdk5 and p35 in the CPu, while no correlation was observed in males. Our studies suggest that repeated cocaine administration induced different patterns of behavioral and molecular responses in the PKA cascade in male and female rats. PMID:27553823

  11. PKA-chromatin association at stress responsive target genes from Saccharomyces cerevisiae.

    PubMed

    Baccarini, Leticia; Martínez-Montañés, Fernando; Rossi, Silvia; Proft, Markus; Portela, Paula

    2015-11-01

    Gene expression regulation by intracellular stimulus-activated protein kinases is essential for cell adaptation to environmental changes. There are three PKA catalytic subunits in Saccharomyces cerevisiae: Tpk1, Tpk2, and Tpk3 and one regulatory subunit: Bcy1. Previously, it has been demonstrated that Tpk1 and Tpk2 are associated with coding regions and promoters of target genes in a carbon source and oxidative stress dependent manner. Here we studied five genes, ALD6, SED1, HSP42, RPS29B, and RPL1B whose expression is regulated by saline stress. We found that PKA catalytic and regulatory subunits are associated with both coding regions and promoters of the analyzed genes in a stress dependent manner. Tpk1 and Tpk2 recruitment was completely abolished in catalytic inactive mutants. BCY1 deletion changed the binding kinetic to chromatin of each Tpk isoform and this strain displayed a deregulated gene expression in response to osmotic stress. In addition, yeast mutants with high PKA activity exhibit sustained association to target genes of chromatin-remodeling complexes such as Snf2-catalytic subunit of the SWI/SNF complex and Arp8-component of INO80 complex, leading to upregulation of gene expression during osmotic stress. Tpk1 accumulation in the nucleus was stimulated upon osmotic stress, while the nuclear localization of Tpk2 and Bcy1 showed no change. We found that each PKA subunit is transported into the nucleus by a different β-karyopherin pathway. Moreover, β-karyopherin mutant strains abolished the chromatin association of Tpk1 or Tpk2, suggesting that nuclear localization of PKA catalytic subunits is required for its association to target genes and properly gene expression.

  12. Nutritional value of 15 corn gluten meals for growing pigs: chemical composition, energy content and amino acid digestibility.

    PubMed

    Ji, Ying; Zuo, Lei; Wang, Fengli; Li, Defa; Lai, Changhua

    2012-08-01

    The objectives of this study were to determine the chemical composition, energy content and amino acid digestibility for corn gluten meals (CGM) and to develop prediction equations for estimating energy content and amino acid digestibility for growing pigs based on the chemical characteristics of these meals. The 15 CGM tested were obtained from seven Chinese companies. Experiment 1 was conducted to determine the digestible (DE) and metabolisable energy (ME) of the 15 CGM. The 18 growing barrows (38 +/- 4 kg) were assigned to three 6 x 6 Latin square designs. The 15 CGM test diets were formulated to contain 19.20% CGM, which replaced 20% of the energy supplied by corn and crystalline amino acid in the basal diet. Experiment 2 was conducted to determine the apparent (AID) and standardised (SID) ileal digestibility of the crude protein (CP) and amino acids in the 15 CGM using chromic oxide as an inert marker. The 18 growing barrows (25 +/- 2 kg) fitted with a simple T-cannula were assigned to three 6 x 6 Latin square designs. The 15 test diets contained 35% of one of the 15 CGM as the sole source of amino acids in the diet. The results showed a considerable variation in the chemical composition of CGM within and among plants. On dry matter basis, the DE and ME content of the CGM ranged from 18.8 to 21.0 MJ/kg and from 18.0 to 19.9 MJ/kg, respectively. There were no significant differences in the AID and SID for CP, arginine, lysine, glycine and proline among the 15 CGM, however, for all the other amino acids, significant differences were found for their AID and SID. With R2 values exceeding 0.50, the DE of CGM can be predicted accurately from CP and fibre content and ME from starch and fibre content. Suitable prediction equations for SID of methionine were also developed.

  13. Recent Progress on the Conversion of Surplus Picric Acid/Explosive D to Higher Value Products

    SciTech Connect

    R.Mitchell, A; Hsu, P C; Coburn, M D; Schmidt, R D; Pagoria, P F; Lee, G S; Kwak, S W

    2004-07-06

    The global demilitarization of nuclear and conventional munitions is producing millions of pounds of surplus energetic materials. Historically, energetic materials (high explosives, propellants, and pyrotechnics) have been disposed of by open burning/open detonation (OB/OD). The use of OB/OD is becoming unacceptable due to public concerns and increasingly stringent environmental regulations. Clearly, there is a great need to develop environmentally sound and cost-effective alternatives to OB/OD. The conversion of surplus picric acid and/or ammonium picrate (Explosive D) to1,3,5-triamino-2,4,6- trinitrobenzene (TATB) has been subject of extensive process development studies at Lawrence Livermore National Laboratory (LLNL). LLNL, under the direction and sponsorship of the U.S. Army Defense Ammunition Center (DAC), is developing a process for the conversion of picric acid to TATB on a larger scale. In FY 03, a 10 g per batch process was developed with good results. Development for a one pound per batch system is required as part of overall scale up process for producing TATB from the surplus feedstocks.

  14. Fatty acid profile, total cholesterol, vitamin content, and TBARS value of turkey breast muscle cured with the addition of lycopene.

    PubMed

    Skiepko, N; Chwastowska-Siwiecka, I; Kondratowicz, J; Mikulski, D

    2016-05-01

    The aim of this study was to determine the effect of lycopene addition for curing turkey meat on the profile of fatty acids, total cholesterol, vitamin content, and the TBARS of the final products. The analyzed material comprised 64 breast muscles, of which 16 (RBM) were immediately transported to a laboratory. Another 16 (UBM) were heat treated in a convection steam oven, and 32 muscles were cured for 3 days in two types of curing mixture: without (CBM) and with (CBM+Lyc) tomato peel extract standardized for 5% lycopene content. After completed curing, samples were steamed and grilled under the same conditions as raw samples. Statistical analysis demonstrated the highest (P≤0.01) mean content of vitamin A (0.07 μg/g) in chilled muscles. The content of vitamin E was lower (P≤0.01) in UBM samples than in CBM+Lyc and RBM. The TBARS value was the lowest (P≤0.01) in RBM muscles (0.35 mg MDA/kg of meat). Although there were no differences between products, but lower TBARS were found in CBM+Lyc samples. The content of cholesterol was higher (P≤0.01) in CBM+Lyc products than in the RBM and UBM. RBM samples contained (P≤0.01) the lowest amount of saturated, monounsaturated, and hypercholesterolemic fatty acids, and the highest of unsaturated, polyunsaturated, and hypocholesterolemic fatty acids. CBM+Lyc samples contained (P≤0.01) less hypercholesterolemic and more hypocholesterolemic fatty acids than CBM group. Higher (P≤0.01) unsaturated/saturated and hypocholesterolemic/hypercholesterolemic fatty acid ratios were also found in CBM+Lyc products. The study demonstrated that the used processing technology caused reduction (P≤0.01) of n-3 and n-6 PUFA content. Findings suggest that the addition of lycopene in the process of meat curing and heat treatment in meat industry do not change the content of vitamins and cholesterol or alter the TBARS value in turkey meat products. Nevertheless, lycopene can be used to increase the content of essential

  15. Lipase-catalyzed process in an anhydrous medium with enzyme reutilization to produce biodiesel with low acid value.

    PubMed

    Azócar, Laura; Ciudad, Gustavo; Heipieper, Hermann J; Muñoz, Robinson; Navia, Rodrigo

    2011-12-01

    One major problem in the lipase-catalyzed production of biodiesel or fatty acid methyl esters (FAME) is the high acidity of the product, mainly caused by water presence, which produces parallel hydrolysis and esterification reactions instead of transesterification to FAME. Therefore, the use of reaction medium in absence of water (anhydrous medium) was investigated in a lipase-catalyzed process to improve FAME yield and final product quality. FAME production catalyzed by Novozym 435 was carried out using waste frying oil (WFO) as raw material, methanol as acyl acceptor, and 3Å molecular sieves to extract the water. The anhydrous conditions allowed the esterification of free fatty acids (FFA) from feedstock at the initial reaction time. However, after the initial esterification process, water absence avoided the consecutives reactions of hydrolysis and esterification, producing FAME mainly by transesterification. Using this anhydrous medium, a decreasing in both the acid value and the diglycerides content in the product were observed, simultaneously improving FAME yield. Enzyme reuse in the anhydrous medium was also studied. The use of the moderate polar solvent tert-butanol as a co-solvent led to a stable catalysis using Novozym 435 even after 17 successive cycles of FAME production under anhydrous conditions. These results indicate that a lipase-catalyzed process in an anhydrous medium coupled with enzyme reuse would be suitable for biodiesel production, promoting the use of oils of different origin as raw materials.

  16. Hydrometallurgical process for the recovery of metal values from spent lithium-ion batteries in citric acid media.

    PubMed

    Chen, Xiangping; Zhou, Tao

    2014-11-01

    In this paper, a hydrometallurgical process has been proposed to recover valuable metals from spent lithium-ion batteries in citric acid media. Leaching efficiencies as high as 97%, 95%, 94%, and 99% of Ni, Co, Mn, and Li were achieved under the optimal leaching experimental conditions of citric acid concentration of 2 mol L(-1), leaching temperature of 80 °C, leaching time of 90 min, liquid-solid ratio of 30 ml g(-1), and 2 vol. % H2O2. For the metals recovery process, nickel and cobalt were selectively precipitated by dimethylglyoxime reagent and ammonium oxalate sequentially. Then manganese was extracted by Na-D2EHPA and the manganese-loaded D2EHPA was stripped with sulfuric acid. The manganese was recovered as MnSO4 in aqueous phase and D2EHPA could be reused after saponification. Finally, lithium was precipitated by 0.5 mol L(-1) sodium phosphate. Under their optimal conditions, the recovery percentages of Ni, Co, Mn, and Li can reach 98%, 97%, 98%, and 89%, respectively. This is a relatively simple route in which all metal values could be effectively leached and recovered in citric acid media.

  17. Value-added potential of expeller-pressed canola oil refining: characterization of sinapic acid derivatives and tocopherols from byproducts.

    PubMed

    Chen, Yougui; Thiyam-Hollander, Usha; Barthet, Veronique J; Aachary, Ayyappan A

    2014-10-01

    Valuable phenolic antioxidants are lost during oil refining, but evaluation of their occurrence in refining byproducts is lacking. Rapeseed and canola oil are both rich sources of sinapic acid derivatives and tocopherols. The retention and loss of sinapic acid derivatives and tocopherols in commercially produced expeller-pressed canola oils subjected to various refining steps and the respective byproducts were investigated. Loss of canolol (3) and tocopherols were observed during bleaching (84.9%) and deodorization (37.6%), respectively. Sinapic acid (2) (42.9 μg/g), sinapine (1) (199 μg/g), and canolol (344 μg/g) were found in the refining byproducts, namely, soap stock, spent bleaching clay, and wash water, for the first time. Tocopherols (3.75 mg/g) and other nonidentified phenolic compounds (2.7 mg sinapic acid equivalent/g) were found in deodistillates, a byproduct of deodorization. DPPH radical scavenging confirmed the antioxidant potential of the byproducts. This study confirms the value-added potential of byproducts of refining as sources of endogenous phenolics.

  18. Hydrometallurgical process for the recovery of metal values from spent lithium-ion batteries in citric acid media.

    PubMed

    Chen, Xiangping; Zhou, Tao

    2014-11-01

    In this paper, a hydrometallurgical process has been proposed to recover valuable metals from spent lithium-ion batteries in citric acid media. Leaching efficiencies as high as 97%, 95%, 94%, and 99% of Ni, Co, Mn, and Li were achieved under the optimal leaching experimental conditions of citric acid concentration of 2 mol L(-1), leaching temperature of 80 °C, leaching time of 90 min, liquid-solid ratio of 30 ml g(-1), and 2 vol. % H2O2. For the metals recovery process, nickel and cobalt were selectively precipitated by dimethylglyoxime reagent and ammonium oxalate sequentially. Then manganese was extracted by Na-D2EHPA and the manganese-loaded D2EHPA was stripped with sulfuric acid. The manganese was recovered as MnSO4 in aqueous phase and D2EHPA could be reused after saponification. Finally, lithium was precipitated by 0.5 mol L(-1) sodium phosphate. Under their optimal conditions, the recovery percentages of Ni, Co, Mn, and Li can reach 98%, 97%, 98%, and 89%, respectively. This is a relatively simple route in which all metal values could be effectively leached and recovered in citric acid media. PMID:25378255

  19. Biomimetic Fenton-catalyzed lignin depolymerization to high-value aromatics and dicarboxylic acids.

    PubMed

    Zeng, Jijiao; Yoo, Chang Geun; Wang, Fei; Pan, Xuejun; Vermerris, Wilfred; Tong, Zhaohui

    2015-03-01

    By mimicking natural lignin degradation systems, the Fenton catalyst (Fe(3+), H2O2) can effectively facilitate lignin depolymerization in supercritical ethanol (7 MPa, 250 °C) to give organic oils that consist of mono- and oligomeric aromatics, phenols, dicarboxylic acids, and their derivatives in yields up to (66.0±8.5) %. The thermal properties, functional groups, and surface chemistry of lignin before and after Fenton treatment were examined by thermogravimetric analysis, pyrolysis-gas chromatography-mass spectrometry, (31)P NMR spectroscopy, and X-ray photoelectron spectroscopy. The results suggest that the Fenton catalyst facilitates lignin depolymerization through cleavage of β-ether bonds between lignin residues. The formation of a lignin-iron chelating complex effectively depresses lignin recondensation; thus minimizing charcoal formation and enhancing the yield of liquid products.

  20. Determination of the acid values of edible oils via FTIR spectroscopy based on the OH stretching band.

    PubMed

    Jiang, Xiuming; Li, Shen; Xiang, Guoqiang; Li, Qiuhong; Fan, Lu; He, Lijun; Gu, Keren

    2016-12-01

    A new method for determining the acid values (AVs) of edible oils based on the OH stretching band was developed. The oil sample was diluted with carbon tetrachloride and was placed in a quartz cuvette with a thickness of 1cm to record the FTIR spectrum. The peak at 3535cm(-1), which corresponds to the OH stretch of the carboxyl group in free fatty acids, together with the peak valley at 3508cm(-1) and the spectral data in the range of 3340-3390cm(-1) were used to determine the AV of the edible oil. The excellent linear relationship between the AVs measured in this work and those measured using a titration method, with a correlation coefficient (R) of 0.9929, indicates that the present procedure can be applied as an alternative to the classic method for determining the AVs of edible oils. PMID:27374571

  1. KCNE variants reveal a critical role of the β subunit carboxyl terminus in PKA-dependent regulation of the IKs potassium channel

    PubMed Central

    Kurokawa, Junko; Bankston, John R.; Kaihara, Asami; Chen, Lei; Furukawa, Tetsushi; Kass, Robert S.

    2009-01-01

    Co-assembly of KCNQ1 with different accessory, or beta, subunits that are members of the KCNE family results in potassium (K+) channels that conduct functionally distinct currents. The alpha subunit KCNQ1 conducts a slowly activated delayed rectifier K+ current (IKs), a major contributor to cardiac repolarization, when co-assembled with KCNE1 and channels that favor the open state when co-assembled with either KCNE2 or KCNE3. In the heart, stimulation of the sympathetic nervous system enhances IKs. A macromolecular signaling complex of the IKs channel including the targeting protein Yotiao coordinates up or downregulation of channel activity by protein kinase A (PKA) phosphorylation and dephosphorylation of molecules in the complex. β-adrenergic receptor mediated IKs upregulation, a functional consequence of PKA phosphorylation of the KCNQ1 amino terminus (N-T), requires co-expression of KCNQ1/Yotiao with KCNE1. Here, we report that co-expression of KCNE2, like KCNE1, confers a functional channel response to KCNQ1 phosphorylation, but co-expression of KCNE3 does not. Amino acid sequence comparison among the KCNE peptides, and KCNE1 truncation experiments, reveal a segment of the predicted intracellular KCNE1 carboxyl terminus (C-T) that is necessary for functional transduction of PKA phosphorylated KCNQ1. Moreover, chimera analysis reveals a region of KCNE1 sufficient to confer cAMP-dependent functional regulation upon the KCNQ1_ KCNE3_Yotiao channel. The property of specific beta subunits to transduce post-translational regulation of alpha subunits of ion channels adds another dimension to our understanding molecular mechanisms underlying the diversity of regulation of native K+ channels. PMID:19077539

  2. A Non-Invasive NMR Method Based on Histidine Imidazoles to Analyze the pH-Modulation of Protein-Nucleic Acid Interfaces.

    PubMed

    Cruz-Gallardo, Isabel; Del Conte, Rebecca; Velázquez-Campoy, Adrián; García-Mauriño, Sofía M; Díaz-Moreno, Irene

    2015-05-11

    A useful (2) J(N-H) coupling-based NMR spectroscopic approach is proposed to unveil, at the molecular level, the contribution of the imidazole groups of histidines from RNA/DNA-binding proteins on the modulation of binding to nucleic acids by pH. Such protonation/deprotonation events have been monitored on the single His96 located at the second RNA/DNA recognition motif (RRM2) of T-cell intracellular antigen-1 (TIA-1) protein. The pKa values of the His96 ionizable groups were substantially higher in the complexes with short U-rich RNA and T-rich DNA oligonucleotides than those of the isolated TIA-1 RRM2. Herein, the methodology applied to determine changes in pKa of histidine side chains upon DNA/RNA binding, gives valuable information to understand the pH effect on multidomain DNA/RNA-binding proteins that shuttle among different cellular compartments.

  3. The effect of terebinth (Pistacia terebinthus L.) coffee addition on the chemical and physical characteristics, colour values, organic acid profiles, mineral compositions and sensory properties of ice creams.

    PubMed

    Yüksel, Arzu Kavaz; Şat, Ihsan Güngör; Yüksel, Mehmet

    2015-12-01

    The aim of this research was to evaluate the effect of terebinth (Pistacia terebinthus L.) coffee addition (0.5, 1 and 2 %) on the chemical and physical properties, colour values, organic acid profiles, mineral contents and sensory characteristics of ice creams. The total solids, fat, titratable acidity, viscosity, first dripping time and complete melting time values, a (*) and b (*) colour properties, citric, lactic, acetic and butyric acid levels and Ca, Cu, Mg, Fe, K, Zn and Na concentrations of ice creams showed an increase with the increment of terebinth coffee amount, while protein, pH, L (*), propionic acid and orotic acid values decreased. However, Al and malic acid were not detected in any of the samples. The overall acceptability scores of the sensory properties showed that the addition of 1 % terebinth coffee to the ice cream was more appreciated by the panellists.

  4. The effect of terebinth (Pistacia terebinthus L.) coffee addition on the chemical and physical characteristics, colour values, organic acid profiles, mineral compositions and sensory properties of ice creams.

    PubMed

    Yüksel, Arzu Kavaz; Şat, Ihsan Güngör; Yüksel, Mehmet

    2015-12-01

    The aim of this research was to evaluate the effect of terebinth (Pistacia terebinthus L.) coffee addition (0.5, 1 and 2 %) on the chemical and physical properties, colour values, organic acid profiles, mineral contents and sensory characteristics of ice creams. The total solids, fat, titratable acidity, viscosity, first dripping time and complete melting time values, a (*) and b (*) colour properties, citric, lactic, acetic and butyric acid levels and Ca, Cu, Mg, Fe, K, Zn and Na concentrations of ice creams showed an increase with the increment of terebinth coffee amount, while protein, pH, L (*), propionic acid and orotic acid values decreased. However, Al and malic acid were not detected in any of the samples. The overall acceptability scores of the sensory properties showed that the addition of 1 % terebinth coffee to the ice cream was more appreciated by the panellists. PMID:26604374

  5. In-vivo measurement of intrauterine gases and acid-base values early in human pregnancy.

    PubMed

    Jauniaux, E; Watson, A; Ozturk, O; Quick, D; Burton, G

    1999-11-01

    A new multiparameter sensor that combines electrochemical and fibre-optic technology was used for continuous in-vivo investigation of pH, carbon dioxide partial pressure (PCO(2)), oxygen partial pressure (PO(2)), bicarbonate concentration (HCO(3)(-)), base excess, and oxygen saturation (O(2)Sat) early in human pregnancy. The sensor was inserted into the amniotic cavity and the placental bed of 16 pregnancies at 10-15 weeks gestation, before termination under general anaesthesia. Amniotic fluid and retroplacental blood from the same site were also aspirated and analysed by means of cartridges and a portable blood gas analyser. Eleven series of measurements were obtained. The variation in measurements over the 5 min of monitoring was acid-base with a sensor is stable and accurate. Such technology will be helpful in improving our understanding of the fetoplacental metabolism in normal and complicated pregnancies. PMID:10548645

  6. Value of Caffeic Acid Phenethyl Ester Pretreatment in Experimental Sepsis Model in Rats

    PubMed Central

    Alici, Ozlem; Kavakli, Havva Sahin; Koca, Cemile; Altintas, Neriman Defne; Aydin, Murat; Alici, Suleyman

    2015-01-01

    Background and Aim. The aim of this study was to determine the actions of caffeic acid phenethyl ester (CAPE) on the changes of endothelin-1 (ET-1) level, tumor necrosis factor- (TNF-) alpha, and oxidative stress parameters such as superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels in experimental sepsis model in rats. Materials and Methods. Twenty-four rats were randomly divided into three experimental groups: sham (group 1), sepsis (group 2), and sepsis + CAPE (group 3), n = 8 each. CAPE was administered (10 µmol/kg) intraperitoneally to group 3 before sepsis induction. Serum ET-1, serum TNF-alpha, tissue SOD activity, and tissue MDA levels were measured in all groups. Results. Pretreatment with CAPE decreased ET-1, TNF-alpha, and MDA levels in sepsis induced rats. Additionally SOD activities were higher in rats pretreated with CAPE after sepsis induction. Conclusion. Our results demonstrate that CAPE may have a beneficial effect on ET and TNF-alpha levels and oxidative stress parameters induced by sepsis in experimental rat models. Therefore treatment with CAPE can be used to avoid devastating effects of sepsis. PMID:25948886

  7. Calculating the iodine value for Italian heavy pig subcutaneous adipose tissue from fatty acid methyl ester profiles.

    PubMed

    Lo Fiego, Domenico Pietro; Minelli, Giovanna; Volpelli, Luisa Antonella; Ulrici, Alessandro; Macchioni, Paolo

    2016-12-01

    In this work, different equations were compared as for their effectiveness in predicting the iodine value (IV), based on fatty acid (FA) composition of subcutaneous adipose tissue of Italian heavy pigs. In particular, six equations were tested: AOCS (1); modified AOCS (2), including all unsaturated FA (UFA); regression models obtained using the stepwise regression procedure as variable selection method, calculated considering only UFA (3) or all the FA (4); regression models obtained using the backward elimination procedure, calculated considering only UFA (5) or all the FA (6). The comparison of the equations performance, estimated using an external test set, showed that the use of regression models led to significant enhancements of prediction accuracy with respect to the AOCS equations. Using both equations 4 and 6, the average paired differences between experimental and predicted IV values were not statistically significant. Therefore, it is possible to use these equations for IV estimation of the subcutaneous adipose tissue of Italian heavy pigs. PMID:27522249

  8. In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability.

    PubMed

    Yamauchi, Fumio; Kamioka, Yuji; Yano, Tetsuya; Matsuda, Michiyuki

    2016-09-15

    Vascular hyperpermeability is a pathological hallmark of cancer. Previous in vitro studies have elucidated roles of various signaling molecules in vascular hyperpermeability; however, the activities of such signaling molecules have not been examined in live tumor tissues for technical reasons. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we examined the activity of protein kinase A (PKA), which maintains endothelial barrier function. The level of PKA activity was significantly lower in the intratumoral endothelial cells than the subcutaneous endothelial cells. PKA activation with a cAMP analogue alleviated the tumor vascular hyperpermeability, suggesting that the low PKA activity in the endothelial cells may be responsible for the tumor-tissue hyperpermeability. Because the vascular endothelial growth factor (VEGF) receptor is a canonical inducer of vascular hyperpermeability and a molecular target of anticancer drugs, we examined the causality between VEGF receptor activity and the PKA activity. Motesanib, a kinase inhibitor for VEGF receptor, activated tumor endothelial PKA and reduced the vascular permeability in the tumor. Conversely, subcutaneous injection of VEGF decreased endothelial PKA activity and induced hyperpermeability of subcutaneous blood vessels. Notably, in cultured human umbilical vascular endothelial cells, VEGF activated PKA rather than decreasing its activity, highlighting the remarkable difference between its actions in vitro and in vivo These data suggested that the VEGF receptor signaling pathway increases vascular permeability, at least in part, by reducing endothelial PKA activity in the live tumor tissue. Cancer Res; 76(18); 5266-76. ©2016 AACR.

  9. Differential role of PKA catalytic subunits in mediating phenotypes caused by knockout of the Carney complex gene Prkar1a.

    PubMed

    Yin, Zhirong; Pringle, Daphne R; Jones, Georgette N; Kelly, Kimberly M; Kirschner, Lawrence S

    2011-10-01

    The Carney complex is an inherited tumor predisposition caused by activation of the cAMP-dependent protein kinase [protein kinase A (PKA)] resulting from mutation of the PKA-regulatory subunit gene PRKAR1A. Myxomas and tumors in cAMP-responsive tissues are cardinal features of this syndrome, which is unsurprising given the important role played by PKA in modulating cell growth and function. Previous studies demonstrated that cardiac-specific knockout of Prkar1a causes embryonic heart failure and myxomatous degeneration in the heart, whereas limited Schwann cell-specific knockout of the gene causes schwannoma formation. In this study, we sought to determine the role of PKA activation in this phenotype by using genetic means to reduce PKA enzymatic activity. To accomplish this goal, we introduced null alleles of the PKA catalytic subunits Prkaca (Ca) or Prkacb (Cb) into the Prkar1a-cardiac knockout (R1a-CKO) or limited Schwann cell knockout (R1a-TEC3KO) line. Heterozygosity for Prkaca rescued the embryonic lethality of the R1a-CKO, although mice had a shorter than normal lifespan and died from cardiac failure with atrial thrombosis. In contrast, heterozygosity for Prkacb only enabled the mice to survive 1 extra day during embryogenesis. Biochemical analysis indicated that reduction of Ca markedly reduced PKA activity in embryonic hearts, whereas reduction of Cb had minimal effects. In R1a-TEC3KO mice, tumorigenesis was completely suppressed by a heterozygosity for Prkaca, and by more than 80% by heterozygosity for Prkacb. These data suggest that both developmental and tumor phenotypes caused by Prkar1a mutation result from excess PKA activity due to PKA-Ca. PMID:21852354

  10. In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability.

    PubMed

    Yamauchi, Fumio; Kamioka, Yuji; Yano, Tetsuya; Matsuda, Michiyuki

    2016-09-15

    Vascular hyperpermeability is a pathological hallmark of cancer. Previous in vitro studies have elucidated roles of various signaling molecules in vascular hyperpermeability; however, the activities of such signaling molecules have not been examined in live tumor tissues for technical reasons. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we examined the activity of protein kinase A (PKA), which maintains endothelial barrier function. The level of PKA activity was significantly lower in the intratumoral endothelial cells than the subcutaneous endothelial cells. PKA activation with a cAMP analogue alleviated the tumor vascular hyperpermeability, suggesting that the low PKA activity in the endothelial cells may be responsible for the tumor-tissue hyperpermeability. Because the vascular endothelial growth factor (VEGF) receptor is a canonical inducer of vascular hyperpermeability and a molecular target of anticancer drugs, we examined the causality between VEGF receptor activity and the PKA activity. Motesanib, a kinase inhibitor for VEGF receptor, activated tumor endothelial PKA and reduced the vascular permeability in the tumor. Conversely, subcutaneous injection of VEGF decreased endothelial PKA activity and induced hyperpermeability of subcutaneous blood vessels. Notably, in cultured human umbilical vascular endothelial cells, VEGF activated PKA rather than decreasing its activity, highlighting the remarkable difference between its actions in vitro and in vivo These data suggested that the VEGF receptor signaling pathway increases vascular permeability, at least in part, by reducing endothelial PKA activity in the live tumor tissue. Cancer Res; 76(18); 5266-76. ©2016 AACR. PMID:27488524

  11. Acidic and basic drugs in medicinal chemistry: a perspective.

    PubMed

    Charifson, Paul S; Walters, W Patrick

    2014-12-11

    The acid/base properties of a molecule are among the most fundamental for drug action. However, they are often overlooked in a prospective design manner unless it has been established that a certain ionization state (e.g., quaternary base or presence of a carboxylic acid) appears to be required for activity. In medicinal chemistry optimization programs it is relatively common to attenuate basicity to circumvent undesired effects such as lack of biological selectivity or safety risks such as hERG or phospholipidosis. However, teams may not prospectively explore a range of carefully chosen compound pKa values as part of an overall chemistry strategy or design hypothesis. This review summarizes the potential advantages and disadvantages of both acidic and basic drugs and provides some new analyses based on recently available public data. PMID:25180901

  12. One-step production of biodiesel from oils with high acid value by activated Mg-Al hydrotalcite nanoparticles.

    PubMed

    Wang, Yi-Tong; Fang, Zhen; Zhang, Fan; Xue, Bao-Jin

    2015-10-01

    Activated Mg-Al hydrotalcite (HT-Ca) nanoparticles (<45 nm) were synthesized by co-precipitation and hydrothermal activation with aqueous Ca(OH)2 solution. They were characterized by various techniques including X-ray diffraction, inductively coupled plasma atomic-emission spectrometer, Brunauer-Emmett-Teller method, scanning electronic microscope-X-ray energy dispersive analysis and temperature programmed desorption method. HT-Ca presented both acidic and basic due to the formation of Mg4Al2(OH)14 · 3H2O, Mg2Al(OH)7 and AlO(OH) nanocrystals to esterify and transesterify oils with high acid value (AV). Under conditions of 5 wt% HT-Ca, 160 °C, 30/1 methanol/oil molar ratio and 4h, 93.4% Jatropha biodiesel yield was obtained at AV of 6.3 mg KOH/g with 4 cycles (biodiesel yield>86%). It was further found that it can resist free fatty acids, and biodiesel yield reached 92.9% from soybean oil with high AV of 12.1. HT-Ca catalyst showed a potential practical application for direct production of biodiesel from oils with high AV without pretreatment.

  13. One-step production of biodiesel from oils with high acid value by activated Mg-Al hydrotalcite nanoparticles.

    PubMed

    Wang, Yi-Tong; Fang, Zhen; Zhang, Fan; Xue, Bao-Jin

    2015-10-01

    Activated Mg-Al hydrotalcite (HT-Ca) nanoparticles (<45 nm) were synthesized by co-precipitation and hydrothermal activation with aqueous Ca(OH)2 solution. They were characterized by various techniques including X-ray diffraction, inductively coupled plasma atomic-emission spectrometer, Brunauer-Emmett-Teller method, scanning electronic microscope-X-ray energy dispersive analysis and temperature programmed desorption method. HT-Ca presented both acidic and basic due to the formation of Mg4Al2(OH)14 · 3H2O, Mg2Al(OH)7 and AlO(OH) nanocrystals to esterify and transesterify oils with high acid value (AV). Under conditions of 5 wt% HT-Ca, 160 °C, 30/1 methanol/oil molar ratio and 4h, 93.4% Jatropha biodiesel yield was obtained at AV of 6.3 mg KOH/g with 4 cycles (biodiesel yield>86%). It was further found that it can resist free fatty acids, and biodiesel yield reached 92.9% from soybean oil with high AV of 12.1. HT-Ca catalyst showed a potential practical application for direct production of biodiesel from oils with high AV without pretreatment. PMID:26117239

  14. Treatment of electronic waste to recover metal values using thermal plasma coupled with acid leaching - A response surface modeling approach

    SciTech Connect

    Rath, Swagat S.; Nayak, Pradeep; Mukherjee, P.S.; Roy Chaudhury, G.; Mishra, B.K.

    2012-03-15

    Highlights: Black-Right-Pointing-Pointer Sentences/phrases were modified. Black-Right-Pointing-Pointer Necessary discussions for different figures were included. Black-Right-Pointing-Pointer More discussion have been included on the flue gas analysis. Black-Right-Pointing-Pointer Queries to both the reviewers have been given. - Abstract: The global crisis of the hazardous electronic waste (E-waste) is on the rise due to increasing usage and disposal of electronic devices. A process was developed to treat E-waste in an environmentally benign process. The process consisted of thermal plasma treatment followed by recovery of metal values through mineral acid leaching. In the thermal process, the E-waste was melted to recover the metal values as a metallic mixture. The metallic mixture was subjected to acid leaching in presence of depolarizer. The leached liquor mainly contained copper as the other elements like Al and Fe were mostly in alloy form as per the XRD and phase diagram studies. Response surface model was used to optimize the conditions for leaching. More than 90% leaching efficiency at room temperature was observed for Cu, Ni and Co with HCl as the solvent, whereas Fe and Al showed less than 40% efficiency.

  15. Quantum chemical studies on solvents for post-combustion carbon dioxide capture: calculation of pKa and carbamate stability of disubstituted piperazines.

    PubMed

    Gangarapu, Satesh; Wierda, Gerben J; Marcelis, Antonius T M; Zuilhof, Han

    2014-06-23

    Piperazine is a widely studied solvent for post-combustion carbon dioxide capture. To investigate the possibilities of further improving this process, the electronic and steric effects of -CH(3), -CH(2)F, -CH(2)OH, -CH(2)NH(2), -COCH3 , and -CN groups of 2,5-disubstituted piperazines on the pKa and carbamate stability towards hydrolysis are investigated by quantum chemical methods. For the calculations, B3LYP, M11L, and spin-component-scaled MP2 (SCS-MP2) methods are used and coupled with the SMD solvation model. The experimental pK(a) values of piperazine, 2-methylpiperazine, and 2,5-dimethylpiperazine agree well with the calculated values. The present study indicates that substitution of -CH(3), -CH(2) NH(2), and -CH(2) OH groups on the 2- and 5-positions of piperazine has a positive impact on the CO(2) absorption capacity by reducing the carbamate stability towards hydrolysis. Furthermore, their higher boiling points, relative to piperazine itself, will lead to a reduction of volatility-related losses. PMID:24782140

  16. pKa prediction of monoprotic small molecules the SMARTS way.

    PubMed

    Lee, Adam C; Yu, Jing-Yu; Crippen, Gordon M

    2008-10-01

    Realizing favorable absorption, distribution, metabolism, elimination, and toxicity profiles is a necessity due to the high attrition rate of lead compounds in drug development today. The ability to accurately predict bioavailability can help save time and money during the screening and optimization processes. As several robust programs already exist for predicting logP, we have turned our attention to the fast and robust prediction of pK(a) for small molecules. Using curated data from the Beilstein Database and Lange's Handbook of Chemistry, we have created a decision tree based on a novel set of SMARTS strings that can accurately predict the pK(a) for monoprotic compounds with R(2) of 0.94 and root mean squared error of 0.68. Leave-some-out (10%) cross-validation achieved Q(2) of 0.91 and root mean squared error of 0.80. PMID:18826209

  17. Modeling mutant phenotypes and oscillatory dynamics in the Saccharomyces cerevisiae cAMP-PKA pathway

    PubMed Central

    2013-01-01

    Background The cyclic AMP-Protein Kinase A (cAMP-PKA) pathway is an evolutionarily conserved signal transduction mechanism that regulates cellular growth and differentiation in animals and fungi. We present a mathematical model that recapitulates the short-term and long-term dynamics of this pathway in the budding yeast, Saccharomyces cerevisiae. Our model is aimed at recapitulating the dynamics of cAMP signaling for wild-type cells as well as single (pde1Δ and pde2Δ) and double (pde1Δpde2Δ) phosphodiesterase mutants. Results Our model focuses on PKA-mediated negative feedback on the activity of phosphodiesterases and the Ras branch of the cAMP-PKA pathway. We show that both of these types of negative feedback are required to reproduce the wild-type signaling behavior that occurs on both short and long time scales, as well as the the observed responses of phosphodiesterase mutants. A novel feature of our model is that, for a wide range of parameters, it predicts that intracellular cAMP concentrations should exhibit decaying oscillatory dynamics in their approach to steady state following glucose stimulation. Experimental measurements of cAMP levels in two genetic backgrounds of S. cerevisiae confirmed the presence of decaying cAMP oscillations as predicted by the model. Conclusions Our model of the cAMP-PKA pathway provides new insights into how yeast respond to alterations in their nutrient environment. Because the model has both predictive and explanatory power it will serve as a foundation for future mathematical and experimental studies of this important signaling network. PMID:23680078

  18. Defect-Related Luminescent Hydroxyapatite-Enhanced Osteogenic Differentiation of Bone Mesenchymal Stem Cells Via an ATP-Induced cAMP/PKA Pathway.

    PubMed

    Wang, Chao; Liu, Dandan; Zhang, Cuimiao; Sun, Jiadong; Feng, Weipei; Liang, Xing-Jie; Wang, Shuxiang; Zhang, Jinchao

    2016-05-11

    Novel defect-related hydroxyapatite (DHAP), which combines the advantages of HAP and defect-related luminescence, has the potential application in tissue engineering and biomedical area, because of its excellent capability of monitoring the osteogenic differentiation and material biodegradation. Although the extracellular mechanism of DHAP minerals and PO4(3-) functioning in osteogenic differentiation has been widely studied, the intracellular molecular mechanism through which PO4(3-) mediates osteogenesis of bone mesenchymal stem cells (BMSCs) is not clear. We examined a previously unknown molecular mechanism through which PO4(3-) promoted osteogenesis of BMSCs with an emphasis on adenosine-triphosphate (ATP)-induced cAMP/PKA pathway. Our studies showed that DHAP could be uptaken into lysosome, in which PO4(3-) was released from DHAP, because of the acid environment of lysosome. The released PO4(3-) interacted with ADP to form ATP, and then degraded into adenosine, an ATP metabolite, which interacted with A2b adenosine receptor to activate the cAMP/PKA pathway, resulting in the high expression of osteogenesis-related genes, such as Runx2, BMP-2, and OCN. These findings first revealed the function of ATP-metabolism in bone physiological homeostasis, which may be developed to cure bone metabolic diseases. PMID:27088570

  19. Caffeine protects against alcohol-induced liver fibrosis by dampening the cAMP/PKA/CREB pathway in rat hepatic stellate cells.

    PubMed

    Wang, Qi; Dai, Xuefei; Yang, Wanzhi; Wang, He; Zhao, Han; Yang, Feng; Yang, Yan; Li, Jun; Lv, Xiongwen

    2015-04-01

    Alcoholic liver fibrosis (ALF) is characterized by hyperplasia of extracellular matrix under long-term alcohol stimulation. Hepatic stellate cell (HSC) activation plays an important role in promoting hepatic fibrogenesis. Caffeine, as the main active component of coffee and tea, was widely consumed in daily life. It was always a thought that caffeine can reduce the probability of suffering from liver diseases. In this study, we attempt to validate the hypothesis that caffeine inhibits activation of HSCs which were isolated from rat ALF model. The rats were gavaged by ethanol to establish ALF model and then treated with different concentrations of caffeine or colchicine. Serum was collected to measure the contents of serum alanine aminotransferase (ALT), aspartate transaminase (AST), hyaluronic acid (HA), laminin (LN), N-terminal peptide of type III procollagen (PIIINP) and type IV collagen (CIV). Then liver tissues were obtained for hematoxylin-eosin staining and Sirius-red staining. Others were treated through liver perfusion to isolate primary rat HSCs. Interestingly, we found that caffeine significantly decreased ALT, AST, HA, LN, PIIINP and CIV levels and reversed liver fibrosis in rat ALF models. Results of immunohistochemistry, real-time PCR and western blot indicated that caffeine could reduce fibrosis and inhibit cAMP/PKA/CREB signal pathway in HSC. Caffeine has a preventive effect on ALF. The mechanism may be interpreted that caffeine inhibits the cAMP/PKA/CREB signal pathway through adenosine A2A receptors in HSC.

  20. A TRPV2–PKA Signaling Module for Transduction of Physical Stimuli in Mast Cells

    PubMed Central

    Stokes, Alexander J.; Shimoda, Lori M.N.; Koblan-Huberson, Murielle; Adra, Chaker N.; Turner, Helen

    2004-01-01

    Cutaneous mast cell responses to physical (thermal, mechanical, or osmotic) stimuli underlie the pathology of physical urticarias. In vitro experiments suggest that mast cells respond directly to these stimuli, implying that a signaling mechanism couples functional responses to physical inputs in mast cells. We asked whether transient receptor potential (vanilloid) (TRPV) cation channels were present and functionally coupled to signaling pathways in mast cells, since expression of this channel subfamily confers sensitivity to thermal, osmotic, and pressure inputs. Transcripts for a range of TRPVs were detected in mast cells, and we report the expression, surface localization, and oligomerization of TRPV2 protein subunits in these cells. We describe the functional coupling of TRPV2 protein to calcium fluxes and proinflammatory degranulation events in mast cells. In addition, we describe a novel protein kinase A (PKA)–dependent signaling module, containing PKA and a putative A kinase adapter protein, Acyl CoA binding domain protein (ACBD)3, that interacts with TRPV2 in mast cells. We propose that regulated phosphorylation by PKA may be a common pathway for TRPV modulation. PMID:15249591

  1. PKA-CREB-BDNF signaling regulated long lasting antidepressant activities of Yueju but not ketamine

    PubMed Central

    Xue, Wenda; Wang, Wei; Gong, Tong; Zhang, Hailou; Tao, Weiwei; Xue, Lihong; Sun, Yan; Wang, Fushun; Chen, Gang

    2016-01-01

    Yueju confers antidepressant effects in a rapid and long-lasting manner, similar to ketamine. CREB (cAMP-response element binding protein) signaling is implicated in depression pathology and antidepressant responses. However, the role of CREB and associated brain derived neurotrophic factor (BDNF) signaling in rapid and long-lasting antidepressant effects remains unclear. Here, we demonstrated that ICR and Kunming strain mice conferred antidepressant responses lasting for 1 and 5 days, respectively, following a single dose of Yueju. One day post Yueju in Kunming but not ICR strain mice, expression of total and phosphorylated CREB, as well as the CREB signaling activator, PKA (protein kinase A) was up-regulated in the hippocampus. Although BDNF gene expression increased at 3 hours in both strains, it remained up-regulated at 1 day only in Kunming mice. Ketamine showed similar strain-dependent behavioral effects. However, blockade of PKA/CREB signaling blunted the antidepressant effects and reversed the up-regulation of BDNF gene expression by Yueju, but not ketamine. Conversely, blockade of mammalian target of rapamycin signaling led to opposite effects. Taken altogether, prolonged transcriptional up-regulation of hippocampal BDNF may account for the stain-dependent enduring antidepressant responses to Yueju and ketamine, but it was mediated via PKA/CREB pathway only for Yueju. PMID:27197752

  2. Nutrient Control of Yeast Gametogenesis Is Mediated by TORC1, PKA and Energy Availability

    PubMed Central

    Weidberg, Hilla; Moretto, Fabien; Spedale, Gianpiero; Amon, Angelika; van Werven, Folkert J.

    2016-01-01

    Cell fate choices are tightly controlled by the interplay between intrinsic and extrinsic signals, and gene regulatory networks. In Saccharomyces cerevisiae, the decision to enter into gametogenesis or sporulation is dictated by mating type and nutrient availability. These signals regulate the expression of the master regulator of gametogenesis, IME1. Here we describe how nutrients control IME1 expression. We find that protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling mediate nutrient regulation of IME1 expression. Inhibiting both pathways is sufficient to induce IME1 expression and complete sporulation in nutrient-rich conditions. Our ability to induce sporulation under nutrient rich conditions allowed us to show that respiration and fermentation are interchangeable energy sources for IME1 transcription. Furthermore, we find that TORC1 can both promote and inhibit gametogenesis. Down-regulation of TORC1 is required to activate IME1. However, complete inactivation of TORC1 inhibits IME1 induction, indicating that an intermediate level of TORC1 signalling is required for entry into sporulation. Finally, we show that the transcriptional repressor Tup1 binds and represses the IME1 promoter when nutrients are ample, but is released from the IME1 promoter when both PKA and TORC1 are inhibited. Collectively our data demonstrate that nutrient control of entry into sporulation is mediated by a combination of energy availability, TORC1 and PKA activities that converge on the IME1 promoter. PMID:27272508

  3. Dietary linoleic acid requirements in the presence of α-linolenic acid are lower than the historical 2 % of energy intake value, study in rats.

    PubMed

    Choque, Benjamin; Catheline, Daniel; Delplanque, Bernadette; Guesnet, Philippe; Legrand, Philippe

    2015-04-14

    Previous studies on rats and human subjects have established that the linoleic acid (LA) requirement is 2 % of the total energy intake (en%), but is obtained in the absence of α-linolenic acid (ALA) and consequently appear to be overestimated. This raises questions since a recent study including ALA has suggested to divide the historical value by four. However, this recent study has remained inconclusive because the animals used were not totally LA-deficient animals. For the first time, the present study was especially designed using physiological and biochemical markers and performed in two steps: (1) to achieve a specific n-6 fatty acid deficiency model using growing male rats fed either a 0 en% from LA/0 en% from ALA (0LA/0ALA), 0LA/0·5ALA or 2LA/0·5ALA diet, born from female rats fed a 0LA/0·5ALA diet; and (2) to refine the required level of LA in the presence of ALA using rats fed either a 0LA/0ALA, 0·5LA/0·5ALA, 1LA/0·5ALA, 1·5LA/0·5ALA diet, born from female rats fed a 0LA/0·5ALA diet. The first step shows that the best LA deficiency model was obtained using rats fed the 0LA/0ALA diet, born from female rats fed the 0LA/0·5ALA diet. The second step demonstrates that in growing rats, LA deficiency was corrected with an intake of 1-1·5 en% from LA and 0·5 en% from ALA. These data suggest that the requirements in humans should be revisited, considering the presence of ALA to set up the recommendation for LA.

  4. Phosphorylation of β-catenin by PKA promotes ATP-induced proliferation of vascular smooth muscle cells

    PubMed Central

    Taurin, Sebastien; Sandbo, Nathan; Yau, Douglas M.; Sethakorn, Nan; Dulin, Nickolai O.

    2012-01-01

    Extracellular ATP stimulates proliferation of vascular smooth muscle cells (VSMC) through activation of G protein-coupled P2Y purinergic receptors. We have previously shown that ATP stimulates a transient activation of protein kinase A (PKA), which, together with the established mitogenic signaling of purinergic receptors, promotes proliferation of VSMC (Hogarth DK, Sandbo N, Taurin S, Kolenko V, Miano JM, Dulin NO. Am J Physiol Cell Physiol 287: C449–C456, 2004). We also have shown that PKA can phosphorylate β-catenin at two novel sites (Ser552 and Ser675) in vitro and in overexpression cell models (Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. J Biol Chem 281: 9971–9976, 2006). β-Catenin promotes cell proliferation by activation of a family of T-cell factor (TCF) transcription factors, which drive the transcription of genes implicated in cell cycle progression including cyclin D1. In the present study, using the phosphospecific antibodies against phospho-Ser552 or phospho-Ser675 sites of β-catenin, we show that ATP can stimulate PKA-dependent phosphorylation of endogenous β-catenin at both of these sites without affecting its expression levels in VSMC. This translates to a PKA-dependent stimulation of TCF transcriptional activity through an increased association of phosphorylated (by PKA) β-catenin with TCF-4. Using the PKA inhibitor PKI or dominant negative TCF-4 mutant, we show that ATP-induced cyclin D1 promoter activation, cyclin D1 protein expression, and proliferation of VSMC are all dependent on PKA and TCF activities. In conclusion, we show a novel mode of regulation of endogenous β-catenin through its phosphorylation by PKA, and we demonstrate the importance of this mechanism for ATP-induced proliferation of VSMC. PMID:18353896

  5. Catalytic actions of alkaline salts in reactions between 1,2,3,4-butanetetracarboxylic acid and cellulose: II. Esterification.

    PubMed

    Ji, Bolin; Tang, Peixin; Yan, Kelu; Sun, Gang

    2015-11-01

    1,2,3,4-Butanetetracarboxylic acid (BTCA) reacts with cellulose in two steps with catalysis of alkaline salts such as sodium hypophosphite: anhydride formation and esterification of anhydride with cellulose. The alkali metal ions were found effective in catalyzing formation of BTCA anhydride in a previous report. In this work, catalytic functions of the alkaline salts in the esterification reaction between BTCA anhydride and cellulose were investigated. Results revealed that acid anions play an important role in the esterification reaction by assisting removal of protons on intermediates and completion of the esterification between cellulose and BTCA. Besides, alkaline salts with lower pKa1 values of the corresponding acids are more effective ones for the reaction since addition of these salts could lead to lower pH values and higher acid anion concentrations in finishing baths. The mechanism explains the results of FTIR and wrinkle recovery angles of the fabrics cured under different temperatures and times.

  6. Phosphorylation of Beet black scorch virus coat protein by PKA is required for assembly and stability of virus particles

    PubMed Central

    Zhao, Xiaofei; Wang, Xiaoling; Dong, Kai; Zhang, Yongliang; Hu, Yue; Zhang, Xin; Chen, Yanmei; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei

    2015-01-01

    Plant virus coat proteins (CPs) play a fundamental role in protection of genomic RNAs, virion assembly, and viral movement. Although phosphorylation of several CPs during virus infection have been reported, little information is available about CP phosphorylation of the spherical RNA plant viruses. Here, we demonstrate that the CP of Beet black scorch virus (BBSV), a member of the genus Necrovirus, can be phosphorylated at threonine-41 (T41) by cAMP-dependent protein kinase (PKA)-like kinase in vivo and in vitro. Mutant viruses containing a T41A non-phosphorylatable alanine substitution, and a T41E glutamic acid substitution to mimic threonine phosphorylation were able to replicate but were unable to move systemically in Nicotiana benthamiana. Interestingly, the T41A and T41E mutants generated unstable 17 nm virus-like particles that failed to package viral genomic (g) RNA, compared with wild-type BBSV with 30 nm virions during viral infection in N. benthamiana. Further analyses showed that the T41 mutations had little effect on the gRNA-binding activity of the CP. Therefore, we propose a model whereby CP phosphorylation plays an essential role in long-distance movement of BBSV that involves formation of stable virions. PMID:26108567

  7. Phosphorylation of Beet black scorch virus coat protein by PKA is required for assembly and stability of virus particles.

    PubMed

    Zhao, Xiaofei; Wang, Xiaoling; Dong, Kai; Zhang, Yongliang; Hu, Yue; Zhang, Xin; Chen, Yanmei; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei

    2015-01-01

    Plant virus coat proteins (CPs) play a fundamental role in protection of genomic RNAs, virion assembly, and viral movement. Although phosphorylation of several CPs during virus infection have been reported, little information is available about CP phosphorylation of the spherical RNA plant viruses. Here, we demonstrate that the CP of Beet black scorch virus (BBSV), a member of the genus Necrovirus, can be phosphorylated at threonine-41 (T41) by cAMP-dependent protein kinase (PKA)-like kinase in vivo and in vitro. Mutant viruses containing a T41A non-phosphorylatable alanine substitution, and a T41E glutamic acid substitution to mimic threonine phosphorylation were able to replicate but were unable to move systemically in Nicotiana benthamiana. Interestingly, the T41A and T41E mutants generated unstable 17 nm virus-like particles that failed to package viral genomic (g) RNA, compared with wild-type BBSV with 30 nm virions during viral infection in N. benthamiana. Further analyses showed that the T41 mutations had little effect on the gRNA-binding activity of the CP. Therefore, we propose a model whereby CP phosphorylation plays an essential role in long-distance movement of BBSV that involves formation of stable virions.

  8. Phosphorylation of Beet black scorch virus coat protein by PKA is required for assembly and stability of virus particles.

    PubMed

    Zhao, Xiaofei; Wang, Xiaoling; Dong, Kai; Zhang, Yongliang; Hu, Yue; Zhang, Xin; Chen, Yanmei; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei

    2015-01-01

    Plant virus coat proteins (CPs) play a fundamental role in protection of genomic RNAs, virion assembly, and viral movement. Although phosphorylation of several CPs during virus infection have been reported, little information is available about CP phosphorylation of the spherical RNA plant viruses. Here, we demonstrate that the CP of Beet black scorch virus (BBSV), a member of the genus Necrovirus, can be phosphorylated at threonine-41 (T41) by cAMP-dependent protein kinase (PKA)-like kinase in vivo and in vitro. Mutant viruses containing a T41A non-phosphorylatable alanine substitution, and a T41E glutamic acid substitution to mimic threonine phosphorylation were able to replicate but were unable to move systemically in Nicotiana benthamiana. Interestingly, the T41A and T41E mutants generated unstable 17 nm virus-like particles that failed to package viral genomic (g) RNA, compared with wild-type BBSV with 30 nm virions during viral infection in N. benthamiana. Further analyses showed that the T41 mutations had little effect on the gRNA-binding activity of the CP. Therefore, we propose a model whereby CP phosphorylation plays an essential role in long-distance movement of BBSV that involves formation of stable virions. PMID:26108567

  9. The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy

    PubMed Central

    Stephan, Joseph S.; Yeh, Yuh-Ying; Ramachandran, Vidhya; Deminoff, Stephen J.; Herman, Paul K.

    2009-01-01

    Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway. PMID:19805182

  10. Compartmentalization role of A-kinase anchoring proteins (AKAPs) in mediating protein kinase A (PKA) signaling and cardiomyocyte hypertrophy.

    PubMed

    Rababa'h, Abeer; Singh, Sonal; Suryavanshi, Santosh V; Altarabsheh, Salah Eldien; Deo, Salil V; McConnell, Bradley K

    2014-12-24

    The Beta-adrenergic receptors (β-ARs) stimulation enhances contractility through protein kinase-A (PKA) substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP) has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα), phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2). Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP) levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure-function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.

  11. Capillary Electrophoresis of Substituted Benzoic Acids

    ERIC Educational Resources Information Center

    Mills, Nancy S.; Spence, John D.; Bushey, Michelle M.

    2005-01-01

    A series of substituted benzoic acids (SBAs) are prepared by students. The pKa shift, a result of the electron-withdrawing or electron-donating characteristics of the subsistent is examined in reference to the electrophoretic migration behavior of benzoic acid.

  12. On the nature of the Brønsted acidic groups on native and functionalized mesoporous siliceous SBA-15 as studied by benzylamine adsorption from solution.

    PubMed

    Rosenholm, Jessica M; Czuryszkiewicz, Teresa; Kleitz, Freddy; Rosenholm, Jarl B; Lindén, Mika

    2007-04-10

    The concentration and Brønsted acidity of surface silanol groups on mesoporous silica (SBA-15) has been studied by following the adsorption of benzylamine, BA, from water as a function of pH. The adsorbed amount of BA from water was compared to the maximum amount of BA that could be adsorbed from cyclohexane. Furthermore, the surface concentration and acidity of carboxylic acid functions on surface-functionalized SBA-15 was also studied, which allowed the relative surface concentration of remaining silanols to be obtained. Two types of silanols can be identified, where about 1/5 of the silanols have a pKa pKa of about 8.2. According to the literature, these two types of silanols can be identified as Q3 and Q2 silanols, respectively, of which the Q3 silanols are more acidic. For the surface-functionalized materials, pKa values close to their respective intrinsic values are found for the carboxylic acid functions. However, irrespective of the method of surface functionalization, 50% or more of the accessible surface groups are silanols. The results thus suggest that the effective charge density is largely controlled by the (de)protonation of the silanol groups even for surface-functionalized mesoporous silica, which render the surface chemistry of the surface-functionalized silicas to be radically different from what simple schematic representations would suggest. The results are suggested to be of importance for reaching a predictive level of understanding for the behavior of mesoporous silica in a range of applications, as many of the foreseen applications for mesoporous silica involves water as the medium.

  13. Retention of ionisable compounds on high-performance liquid chromatography XIX. pH variation in mobile phases containing formic acid, piperazine and tris as buffering systems and methanol as organic modifier.

    PubMed

    Subirats, Xavier; Bosch, Elisabeth; Rosés, Martí

    2009-07-10

    In previous works a model to estimate the pH of methanol-aqueous buffer mobile phases from the aqueous pH and concentration of the buffer and the fraction of organic modifier was developed. This model was successfully applied and validated for buffers prepared from ammonia, acetic, phosphoric and citric acids. In the present communication this model has been extended to formic acid, piperazine and tris(hydroxymethyl)aminomethane buffers. Prior to the modelling work, the pK(a) values of the studied buffers at several methanol-water compositions were determined.

  14. The Value of Tranexamic Acid in Reducing Blood Loss following Hip Reconstruction in Children with Cerebral Palsy

    PubMed Central

    Majid, I.; Alshryda, S.; Somanchi, B.; Morakis, E.; Foster, A.

    2015-01-01

    This is a retrospective study of 51 consecutive hip reconstructions in children with cerebral palsy performed between 2011 and 2013. Tranexamic acid (TXA) was used in 14 hip reconstructions only. Transfusion rate was higher, postoperative Hb was lower, and patients stayed longer in the TXA group. This did not reach a statistical significance (P = 0.75, 0.5, and 0.71, resp.). More than half of the patients who had TXA underwent bilateral hip reconstructions in comparison with 27% only in the non-TXA group. Bilateral hip reconstructions mean more surgery, more blood loss, and more blood transfusion. The patients who had TXA were significantly more disabled as evident by the higher proportions of patient with worse GMFCS levels. Although we have not been able to demonstrate the value of TXA in reducing blood loss and transfusion rate in children with CP who underwent hip reconstruction, it is hoped that an interest in exploring the value of TXA in paediatric orthopaedic surgery is generated. Ideally this should be explored further in an adequately powered, randomised controlled trial where risk of bias is minimized. PMID:26664830

  15. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

    PubMed

    Stanger, Simone J; Law, Estelle A; Jamsai, Duangporn; O'Bryan, Moira K; Nixon, Brett; McLaughlin, Eileen A; Aitken, R John; Roman, Shaun D

    2016-08-01

    Spermatozoa require the process of capacitation to enable them to fertilize an egg. PKA is crucial to capacitation and the development of hyperactivated motility. Sperm PKA is activated by cAMP generated by the germ cell-enriched adenylyl cyclase encoded by Adcy10 Male mice lacking Adcy10 are sterile, because their spermatozoa are immotile. The current study was designed to identify binding partners of the sperm-specific (Cα2) catalytic subunit of PKA (PRKACA) by using it as the "bait" in a yeast 2-hybrid system. This approach was used to identify a novel germ cell-enriched protein, sperm PKA interacting factor (SPIF), in 25% of the positive clones. Homozygous Spif-null mice were embryonically lethal. SPIF was coexpressed and coregulated with PRKACA and with t-complex protein (TCP)-11, a protein associated with PKA signaling. We established that these 3 proteins form part of a novel complex in mouse spermatozoa. Upon capacitation, the SPIF protein becomes tyrosine phosphorylated in >95% of sperm. An apparent molecular rearrangement in the complex occurs, bringing PRKACA and TCP11 into proximity. Taken together, these results suggest a role for the novel complex of SPIF, PRKACA, and TCP11 during sperm capacitation, fertilization, and embryogenesis.-Stanger, S. J., Law, E. A., Jamsai, D., O'Bryan, M. K., Nixon, B., McLaughlin, E. A., Aitken, R. J., Roman, S. D. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

  16. Seasonal and spatial changes of free and bound organic acids in total suspended particles in Guangzhou, China

    NASA Astrophysics Data System (ADS)

    Ma, Shexia; Peng, Ping'an; Song, Jianzhong; Bi, Xinhui; Zhao, Jinping; He, Lulu; Sheng, Guoying; Fu, Jiamo

    2010-12-01

    The concentrations and compositions of free and bound organic acids in total suspended particles from typical urban, suburban and forest park sites of Guangzhou were determined in this study. The free form of organic acids (solvent extractable) in aerosols in Guangzhou varied with site and season. The suburban samples contained the highest contents of alkanoic, alkenoic and dicarboxylic acids. These findings were consistent with a higher supply of hydrocarbons and NOx in the suburban area. However, concentrations of aromatic acids were similar in the urban, suburban and forest park sites. Generally, winter season samples of the acids from anthropogenic sources contained more organic acids than summer season samples due to stronger removal by wet deposition in the summer. For the acids from botanic sources, the summer season samples were higher. In addition to the free acids, bound acids (solvent non-extractable) mainly formed by esterification of free acids were also found in the samples. In general, bound acids were higher than free acids. Esterification is mainly controlled by the pKa of organic acids and the atmospheric pH value. This explains why aromatic and dicarboxylic acids occur mainly as bound forms and why the samples from urban sites contained high levels of bound acids as the pH of rain water can reach 4.53. Concentrations of alkanoic and alkenoic acids in the aerosols of Guangzhou were much higher than those in the other areas studied.

  17. Influence of amino acid replacement at position 198 on catalytic properties of zinc-bound water in human carbonic anhydrase III.

    PubMed

    LoGrasso, P V; Tu, C; Chen, X; Taoka, S; Laipis, P J; Silverman, D N

    1993-06-01

    Carbonic anhydrase III, found predominantly in skeletal muscle, is the least efficient of the mammalian carbonic anhydrases in catalyzing the hydration of CO2. Phenylalanine-198 is located on the hydrophobic side of the active-site cavity with its phenyl ring in the proximity of the catalytically active zinc-bound water. We replaced phenylalanine-198 in human carbonic anhydrase III with seven other amino acids (Ala, Asn, Asp, His, Leu, Tyr, Val) using site-directed mutagenesis. The catalytic properties of these enzymes were determined by stopped-flow spectrophotometry, and the exchange of 18O between CO2 and water was measured by mass spectrometry. All of the mutants had maximal values of kcat/Km for the hydration of CO2 enhanced, and five of the mutants had the pKa of the zinc-bound water increased compared with the wild-type enzyme. The largest effects were observed with the replacement Phe-198-->Asp which increased the maximal kcat/Km 140-fold and increased the pKa of the zinc-bound water from near 5 to 9.2. A Brønsted correlation was observed between log(kcat/Km) for hydration of CO2 and the pKa of the zinc-bound water (correlation coefficient r = 0.92); in addition, this pKa was inversely correlated with hydrophobicity of the residue at 198 (correlation coefficient r = -0.83). A direct correlation between the logarithm of the maximal kcat/Km for hydration and the logarithm of the pH-independent value of Ki for inhibition by cyanate (r = 0.95) indicated that the effect of the mutations at residue 198 occurred in large part by enhancement of the rate of dissociation of the enzyme-bicarbonate complex. PMID:8504098

  18. How Ca2+-permeable AMPA receptors, the kinase PKA, and the phosphatase PP2B are intertwined in synaptic LTP and LTD.

    PubMed

    Hell, Johannes W

    2016-01-01

    Both synaptic long-term potentiation (LTP) and long-term depression (LTD) are thought to be critical for memory formation. Dell'Acqua and co-workers now demonstrate that transient postsynaptic incorporation of Ca(2+)-permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is required for LTD in the exemplary hippocampal CA1 region in 2-week-old mice. Mechanistically, LTD depends on AKAP150-anchored protein kinase A (PKA) to promote the initial functional recruitment of CP-AMPARs during LTD induction and on AKAP150-anchored protein phosphatase 2B (PP2B) to trigger their subsequent removal as part of the lasting depression of synaptic transmission. PMID:27117250

  19. How Ca2+-permeable AMPA receptors, the kinase PKA, and the phosphatase PP2B are intertwined in synaptic LTP and LTD.

    PubMed

    Hell, Johannes W

    2016-04-26

    Both synaptic long-term potentiation (LTP) and long-term depression (LTD) are thought to be critical for memory formation. Dell'Acqua and co-workers now demonstrate that transient postsynaptic incorporation of Ca(2+)-permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is required for LTD in the exemplary hippocampal CA1 region in 2-week-old mice. Mechanistically, LTD depends on AKAP150-anchored protein kinase A (PKA) to promote the initial functional recruitment of CP-AMPARs during LTD induction and on AKAP150-anchored protein phosphatase 2B (PP2B) to trigger their subsequent removal as part of the lasting depression of synaptic transmission.

  20. Resolving the bulk δ 15N values of ancient human and animal bone collagen via compound-specific nitrogen isotope analysis of constituent amino acids

    NASA Astrophysics Data System (ADS)

    Styring, Amy K.; Sealy, Judith C.; Evershed, Richard P.

    2010-01-01

    Stable nitrogen isotope analysis is a fundamental tool in assessing dietary preferences and trophic positions within contemporary and ancient ecosystems. In order to assess more fully the dietary contributions to human tissue isotope values, a greater understanding of the complex biochemical and physiological factors which underpin bulk collagen δ 15N values is necessary. Determinations of δ 15N values of the individual amino acids which constitute bone collagen are necessary to unravel these relationships, since different amino acids display different δ 15N values according to their biosynthetic origins. A range of collagen isolates from archaeological faunal and human bone ( n = 12 and 11, respectively), representing a spectrum of terrestrial and marine protein origins and diets, were selected from coastal and near-coastal sites at the south-western tip of Africa. The collagens were hydrolysed and δ 15N values of their constituent amino acids determined as N-acetylmethyl esters (NACME) via gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The analytical approach employed accounts for 56% of bone collagen nitrogen. Reconstruction of bulk bone collagen δ 15N values reveals a 2‰ offset from bulk collagen δ 15N values which is attributable to the δ 15N value of the amino acids which cannot currently be determined by GC-C-IRMS, notably arginine which comprises 53% of the nitrogen unaccounted for (23% of the total nitrogen). The δ 15N values of individual amino acids provide insights into both the contributions of various amino acids to the bulk δ 15N value of collagen and the factors influencing trophic position and the nitrogen source at the base of the food web. The similarity in the δ 15N values of alanine, glutamate, proline and hydroxyproline reflects the common origin of their amino groups from glutamate. The depletion in the δ 15N value of threonine with increasing trophic level indicates a fundamental difference between

  1. Gabapentin activates ROMK1 channels by a protein kinase A (PKA)-dependent mechanism

    PubMed Central

    Lee, C-H; Tsai, T-S; Liou, H-H

    2008-01-01

    Background and purpose: Gabapentin is an effective anticonvulsant. The major physiological function of renal outer medullary potassium (ROMK1) channels is to maintain the resting membrane potential (RMP). We investigated the effect of gabapentin on ROMK1 channels and the mechanism involved. Experimental approach: Xenopus oocytes were injected with mRNA coding for wild-type or mutant ROMK1 channels and giant inside-out patch-clamp recordings were performed. Key results: Gabapentin increased the activity of ROMK1 channels, concentration-dependently and enhanced the activity of wild-type and an intracellular pH (pHi)-gating residue mutant (K80M) channels over a range of pHi. Gabapentin also increased activity of channels mutated at phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sites (R188Q, R217A and K218A). However, gabapentin failed to enhance channel activity in the presence of protein kinase A (PKA) inhibitors and did not activate phosphorylation site mutants (S44A, S219A or S313A), mutants that mimicked the negative charge carried by a phosphate group bound to a serine (S44D, S219D or S313D), or a mutated channel with a positive charge (S219R). These findings show that gabapentin activates ROMK1 channels independently of the pHi and not via a PIP2-dependent pathway. The effects of gabapentin on ROMK1 channels may be due to a PKA-mediated phosphorylation-induced conformational change, but not to charge–charge interactions. Conclusions and implications: ROMK1 channels are the main channels responsible for maintaining the RMP during cellular excitation. Gabapentin increased the activity of ROMK1 channels by a PKA-dependent mechanism, reducing neuronal excitability, and this may play an important role in its antiepileptic effect. PMID:18311184

  2. The effect of pK(a) on pyrimidine/pyridine-derived histamine H4 ligands.

    PubMed

    Savall, Brad M; Meduna, Steven P; Venable, Jennifer; Wei, Jianmei; Smith, Russell C; Hack, Michael D; Thurmond, Robin L; McGovern, Patricia; Edwards, James P

    2014-12-01

    During the course of our efforts toward the discovery of human histamine H4 antagonists from a series of 2-aminiopyrimidines, it was noted that a 6-trifluoromethyl group dramatically reduced affinity of the series toward the histamine H4 receptor. This observation was further investigated by synthesizing a series of ligands that varied in pKa of the pyrimidine derived H4 ligands by over five orders of magnitude and the effect on histamine H4 affinity. This trend was then extended to the discovery of C-linked piperidinyl-2-amino pyridines as histamine H4 receptor antagonists.

  3. Wnt Signaling Inhibits Osteoclast Differentiation by Activating Canonical and Noncanonical cAMP/PKA Pathways

    PubMed Central

    Weivoda, Megan M; Ruan, Ming; Hachfeld, Christine M; Pederson, Larry; Howe, Alan; Davey, Rachel A; Zajac, Jeffrey D; Kobayashi, Yasuhiro; Williams, Bart O; Westendorf, Jennifer J; Khosla, Sundeep; Oursler, Merry Jo

    2016-01-01

    Although there has been extensive characterization of the Wnt signaling pathway in the osteoblast lineage, the effects of Wnt proteins on the osteoclast lineage are less well studied. We found that osteoclast lineage cells express canonical Wnt receptors. Wnt3a reduced osteoclast formation when applied to early bone-marrow macrophage (BMM) osteoclast differentiation cultures, whereas late addition did not suppress osteoclast formation. Early Wnt3a treatment inactivated the crucial transcription factor NFATc1 in osteoclast progenitors. Wnt3a led to the accumulation of nuclear β-catenin, confirming activation of canonical Wnt signaling. Reducing low-density lipoprotein receptor-related proteins (Lrp) 5 and Lrp6 protein expression prevented Wnt3a-induced inactivation of NFATc1; however, deletion of β-catenin did not block Wnt3a inactivation of NFATc1, suggesting that this effect was mediated by a noncanonical pathway. Wnt3a rapidly activated the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and pharmacological stimulation of cAMP/PKA signaling suppressed osteoclast differentiation; Wnt3a-induced NFATc1 phosphorylation was blocked by inhibiting interactions between PKA and A-kinase anchoring proteins (AKAPs). These data indicate that Wnt3a directly suppresses osteoclast differentiation through both canonical (β-catenin) and noncanonical (cAMP/PKA) pathways in osteoclast precursors. In vivo reduction of Lrp5 and Lrp6 expressions in the early osteoclast lineage via Rank promoter Cre recombination reduced trabecular bone mass, whereas disruption of Lrp5/6 expression in late osteoclast precursors via cathepsin K (Ctsk) promoter Cre recombination did not alter the skeletal phenotype. Surprisingly, reduction of Lrp5/6 in the early osteoclast lineage decreased osteoclast numbers, as well as osteoblast numbers. Published studies have previously noted that β-catenin signaling is required for osteoclast progenitor proliferation. Our in vivo data

  4. Transcriptional profiling of immortalized and K-ras-transformed mouse fibroblasts upon PKA stimulation by forskolin in low glucose availability.

    PubMed

    Chiaradonna, Ferdinando; Pirola, Yuri; Ricciardiello, Francesca; Palorini, Roberta

    2016-09-01

    Forskolin (FSK) induces activation of protein kinase A (PKA). This activation protects specifically some cancer cells from death induced by glucose starvation. Cell effects upon FSK treatment prompted us to investigate in detail the physiological role of PKA in the activation of pro-survival mechanisms in glucose starvation. In this regard we performed a microarray analysis of normal NIH3T3 and transformed NIH3T3-K-ras mouse fibroblasts cultured at 1 mM glucose and daily treated or not with 10 μM FSK until 72 h of growth, when the samples were collected. The microarray is deposited into Gene Expression Omnibus under Series GSE68266. The microarray data revealed that the activation of PKA regulates the expression of genes involved in metabolic, stress-response and pro-survival processes, like glutamine metabolism, autophagy and unfolded protein response, preventing cancer cell death in glucose starvation. Altogether these findings suggest that PKA activation, by inducing a complex transcriptional program, leads to cancer survival in nutrient stress, a typical feature of developing tumor. These transcriptional data, identifying this important role of PKA, will be useful to identify novel target in cancer therapy. PMID:27486565

  5. [Clinical value of the sequential study of the uric acid in CSF in patients with cerebral diseases: Part I. Brain tumor and the effect of irradiation].

    PubMed

    Ueda, T; Wakisaka, S; Kinoshita, K; Adachi, H

    1984-03-01

    Uric acid is the end product of the purine metabolism in the human and is mainly excreted to the urine. The studies on cerebrospinal fluid (CSF) uric acid in patients with various neurological diseases were reported in the literature. In the present study the authors discussed the clinical value of the sequential study of the CSF uric acid content in patients with brain tumors. CSF was investigated for uric acid in 23 controls and 30 cases of brain tumor. The results were as follows: The mean value and standard deviation of the uric acid in CSF in controls was 0.23 +/- 0.13 mg/dl. The uric acid in CSF increased in patients with malignant brain tumor (0.49 +/- 0.22 mg/dl, p less than 0.005), but was in normal range in patients with benign brain tumor (0.32 +/- 0.13 mg/dl, 0.10 less than p less than 0.25). There was no significant correlation between CSF uric acid and CSF protein contents. Uric acid in the lumbar CSF was approximately 4 times higher than in the ventricular CSF in patients with brain tumor. The CSF uric acid had progressively increased during irradiation to the whole brain. The factors contributing to increase of the uric acid in CSF were thought to be increased permeability of blood-CSF barrier, global damage of brain tissue, increased nucleic acid catabolism in the central nervous system (CNS) for example in tumor, inflammation or immunoreaction, increased of xanthine, hypoxanthine or xanthine oxidase activity in the CNS, directly increased of plasma components into the CSF due to such as subarachnoid hemorrhage, intraventricular hemorrhage, bleeding in the tumor or surgical operation, dysfunction of the CSF dynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Adding explicit solvent molecules to continuum solvent calculations for the calculation of aqueous acid dissociation constants.

    PubMed

    Kelly, Casey P; Cramer, Christopher J; Truhlar, Donald G

    2006-02-23

    Aqueous acid dissociation free energies for a diverse set of 57 monoprotic acids have been calculated using a combination of experimental and calculated gas and liquid-phase free energies. For ionic species, aqueous solvation free energies were calculated using the recently developed SM6 continuum solvation model. This model combines a dielectric continuum with atomic surface tensions to account for bulk solvent effects. For some of the acids studied, a combined approach that involves attaching a single explicit water molecule to the conjugate base (anion), and then surrounding the resulting anion-water cluster by a dielectric continuum, significantly improves the agreement between the calculated pK(a) value and experiment. This suggests that for some anions, particularly those concentrating charge on a single exposed heteroatom, augmenting implicit solvent calculations with a single explicit water molecule is required, and adequate, to account for strong short-range hydrogen bonding interactions between the anion and the solvent. We also demonstrate the effect of adding several explicit waters by calculating the pK(a) of bicarbonate (HCO(3)(-)) using as the conjugate base carbonate (CO(3)(2-)) bound by up to three explicit water molecules.

  7. Structure of D-AKAP2:PKA RI Complex: Insights into AKAP Specificity and Selectivity

    SciTech Connect

    Sarma, Ganapathy N.; Kinderman, Francis S.; Kim, Choel; von Daake, Sventja; Chen, Lirong; Wang, Bi-Cheng; Taylor, Susan S.

    2010-11-22

    A-kinase anchoring proteins (AKAPs) regulate cyclic AMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP 2 (D-AKAP2) binds to the dimerization/docking (D/D) domain of both RI and RII regulatory subunits of PKA with high affinity. Here we have determined the structures of the RI{alpha} D/D domain alone and in complex with D-AKAP2. The D/D domain presents an extensive surface for binding through a well-formed N-terminal helix, and this surface restricts the diversity of AKAPs that can interact. The structures also underscore the importance of a redox-sensitive disulfide in affecting AKAP binding. An unexpected shift in the helical register of D-AKAP2 compared to the RII{alpha}:D-AKAP2 complex structure makes the mode of binding to RI{alpha} novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity.

  8. Different expression of protein kinase A (PKA) regulatory subunits in normal and neoplastic thyroid tissues.

    PubMed

    Ferrero, Stefano; Vaira, Valentina; Del Gobbo, Alessandro; Vicentini, Leonardo; Bosari, Silvano; Beck-Peccoz, Paolo; Mantovani, Giovanna; Spada, Anna; Lania, Andrea G

    2015-04-01

    The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in both cell growth and cell differentiation control. Mutations of the PRKAR1A gene have been found in patients with Carney complex and in a minority of sporadic anaplastic thyroid carcinomas. The aim of the study was to retrospectively evaluate the expression of different PKA regulatory subunits in benign and non benign human thyroid tumours and to correlate their expression with clinical phenotype. Immunohistochemistry demonstrated a significant increase in PRKAR2B expression in both differentiated and undifferentiated (anaplastic) thyroid tumors in comparison with normal thyroid tissues. Conversely, a significant increase in PRKAR1A expression was only demonstrated in undifferentiated thyroid carcinomas in comparison with normal thyroid tissue and differentiated thyroid tumors. In thyroid cancers without lymph nodal metastases PRKAR1A expression was higher in tumours of more than 2 cm in size (T2 and T3) compared to smaller ones (T1). In conclusion, our data shows that an increased PRKAR1A expression is associated with aggressive and undifferentiated thyroid tumors. PMID:25393625

  9. PBR, StAR, and PKA: partners in cholesterol transport in steroidogenic cells.

    PubMed

    Hauet, T; Liu, J; Li, H; Gazouli, M; Culty, M; Papadopoulos, V

    2002-11-01

    Acute stimulation of cholesterol transport into mitochondria involves the cAMP-dependent protein kinase (PKA), peripheral-type benzodiazepine receptor (PBR), and the steroidogenesis acute regulatory (StAR) proteins. We investigated the respective role of these proteins in hormone-induced steroidogenesis. Oligonucleotides antisense, but not sense, to PBR and StAR reduced their respective levels in steroidogenic cells and inhibited hormone-stimulated steroid formation in MA-10 mouse Leydig tumor cells. In search of the proteins regulating PBR we identified a protein, PAP7, which interacts with PBR and the PKA regulatory subunit RIalpha, is present in adrenal and gonadal cells and is found in mitochondria. Overexpression of the full length PAP7 increased the hormone-induced steroid production. However, inhibition of PAP7 expression reduced the gonadotropin-induced steroid formation. In search of a PBR functional antagonist that would facilitate the studies on the biological function of PBR, we screened a phage display library. A 7-mer competitive PBR peptide antagonist was identified, which when transduced into Leydig cells inhibited the benzodiazepine and hormone-stimulated steroid production suggesting that the endogenous PBR agonist/receptor interaction is critical for the hormone-dependent steroidogenesis. These data indicate that hormone-induced cholesterol transport and the subsequent steroid formation is a dynamic multistep process involving protein-protein interactions.

  10. Epac, not PKA catalytic subunit, is required for 3T3-L1 preadipocyte differentiation

    PubMed Central

    Ji, Zhenyu; Mei, Fang C.; Cheng, Xiaodong

    2009-01-01

    Cyclic AMP plays a critical role in adipocyte differentiation and maturation. However, it is not clear which of the two intracellular cAMP receptors, exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor or protein kinase A/cAMP-dependent protein kinase, is essential for cAMP-mediated adipocyte differentiation. In this study, we utilized a well-defined adipose differentiation model system, the murine preadipocyte line 3T3-L1, to address this issue. We showed that knocking down Epac expression in 3T3-L1 cells using lentiviral based small hairpin RNAs down-regulated peroxisome proliferator-activated receptor gamma expression and dramatically inhibited adipogenic conversion of 3T3-L1 cells while inhibiting PKA catalytic subunit activity by two mechanistically distinct inhibitors, heat stable protein kinase inhibitor and H89, had no effect on 3T3-L1 adipocyte differentiation. Moreover, cAMP analog selectively activating Epac was not able to stimulate adipogenic conversion. Our study demonstrated that while PKA catalytic activity is dispensable, activation of Epac is necessary but not sufficient for adipogenic conversion of 3T3-L1 cells. PMID:20036887

  11. Association between PKA gene polymorphism and NTDs in high risk Chinese population in Shanxi

    PubMed Central

    Wu, Jian; Lu, Xiaolin; Wang, Zhen; Shangguan, Shaofang; Chang, Shaoyan; Li, Rui; Wu, Lihua; Bao, Yihua; Niu, Bo; Wang, Li; Zhang, Ting

    2013-01-01

    Objective: This study aimed to investigate the single nucleotide polymorphisms (SNPs) of PKA and neural tube defects (NTDs) in Chinese population. Method: A total of 183 NTDs cases and 200 healthy controls were used in this study. 7 selected single nucleotide polymorphism (SNP) sites in the PKA gene were analyzed with MassArray high-throughput DNA analyzer with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. A series of statistical methods were carried out to investigate the correlation between the SNPs and the patient susceptibility to NTDs. Results: Statistical analysis showed a significant correlation between the SNP sites rs12132032 in PRKACB and NTDs. The AA genotype, A-allele and dominant AA in rs12132032 significantly increased the incidence of NTDs especially anencephaly (OR=3.87, 95% CI: 1.80-8.34 with genotype; OR=2.08, 95% CI: 1.43-3.04 with allele; OR=3.10, 95% CI: 1.53-6.26 with dominant). The T-allele of rs594631 in PRKACB was correlative with NTDs in male but not in female. Conclusions: The gene polymorphism loci rs12132032 in PRKACB maybe a potential risk factor for anencephaly in Chinese population from Shanxi, while gender susceptibility may influence the correlation. PMID:24294386

  12. Soybean diet improves insulin secretion through activation of cAMP/PKA pathway in rats.

    PubMed

    Veloso, Roberto V; Latorraca, Márcia Q; Arantes, Vanessa C; Reis, Marise A B; Ferreira, Fabiano; Boschero, Antonio C; Carneiro, Everardo M

    2008-11-01

    Maternal malnutrition leads to permanent alterations in insulin secretion of offspring and the soybean diet contributes to improve insulin release. At least a soy component, genistein, seems to increase the insulin secretion by activating the cAMP/PKA and PLC/PKC pathways. Here, we investigated the effect of the soybean diet on the expression of PKAalpha and PKCalpha, and insulin secretion in response to glucose and activators of adenylate cyclase and PKC in adult pancreatic rat islets. Rats from mothers fed with 17% or 6% protein (casein) during pregnancy and lactation were maintained with 17% casein (CC and CR groups) or soybean (SC and SR groups) diet until 90 days of life. The soybean diet improved the insulin response to a physiological concentration of glucose in control islets, but only in the presence of supra-physiological concentrations of glucose in islets from CR and SR groups. PMA also improved the insulin response in islets of SC and SR groups. The expression of PKCalpha was similar in all groups. Forskolin increased the insulin secretion; however, the magnitude of the increment was lower in islets from CR and SR groups than in control animals and in those from rats maintained with soybean diet than in rats fed with casein diet. The PKAalpha expression was similar between SR and CR groups and lower in SC than in CC islets. Thus, soybean diet improved the secretory pattern of beta cells, at least in part, by activating the cAMP/PKA-signaling cascade. PMID:18430554

  13. Prostaglandin E2 inhibits tumor necrosis factor-alpha RNA through PKA type I.

    PubMed

    Stafford, Jennifer B; Marnett, Lawrence J

    2008-02-01

    Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that may contribute to the pathogenesis of septic shock, rheumatoid arthritis, cancer, and diabetes. Prostaglandins endogenously produced by macrophages act in an autocrine fashion to limit TNF-alpha production. We investigated the timing and signaling pathway of prostaglandin-mediated inhibition of TNF-alpha production in Raw 264.7 and J774 macrophages. TNF-alpha mRNA levels were rapidly modulated by PGE(2) or carbaprostacylin. PGE(2) or carbaprostacyclin prevented and rapidly terminated on-going TNF-alpha gene transcription within 15 min of prostaglandin treatment. Selective activation of PKA type I, but not PKA type II or Epac, with chemical analogs of cAMP was sufficient to inhibit LPS-induced TNF-alpha mRNA levels. The mechanisms by which prostaglandins limit TNF-alpha mRNA levels may underlie endogenous regulatory mechanisms that limit inflammation, and may have important implications for understanding chronic inflammatory disease pathogenesis. PMID:18060853

  14. STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs

    PubMed Central

    Garcia-Alvarez, Gisela; Lu, Bo; Yap, Kenrick An Fu; Wong, Loo Chin; Thevathasan, Jervis Vermal; Lim, Lynette; Ji, Fang; Tan, Kia Wee; Mancuso, James J.; Tang, Willcyn; Poon, Shou Yu; Augustine, George J.; Fivaz, Marc

    2015-01-01

    STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca2+ entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER–plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites. PMID:25609091

  15. Acidity of the amidoxime functional group in aqueous solution. A combined experimental and computational study

    DOE PAGES

    Mehio, Nada; Lashely, Mark A.; Nugent, Joseph W.; Tucker, Lyndsay; Correia, Bruna; Do-Thanh, Chi-Linh; Dai, Sheng; Hancock, Robert D.; Bryantsev, Vyacheslav S.

    2015-01-26

    Poly(acrylamidoxime) adsorbents are often invoked in discussions of mining uranium from seawater. It has been demonstrated repeatedly in the literature that the success of these materials is due to the amidoxime functional group. While the amidoxime-uranyl chelation mode has been established, a number of essential binding constants remain unclear. This is largely due to the wide range of conflicting pKa values that have been reported for the amidoxime functional group in the literature. To resolve this existing controversy we investigated the pKa values of the amidoxime functional group using a combination of experimental and computational methods. Experimentally, we used spectroscopicmore » titrations to measure the pKa values of representative amidoximes, acetamidoxime and benzamidoxime. Computationally, we report on the performance of several protocols for predicting the pKa values of aqueous oxoacids. Calculations carried out at the MP2 or M06-2X levels of theory combined with solvent effects calculated using the SMD model provide the best overall performance with a mean absolute error of 0.33 pKa units and 0.35 pKa units, respectively, and a root mean square deviation of 0.46 pKa units and 0.45 pKa units, respectively. Finally, we employ our two best methods to predict the pKa values of promising, uncharacterized amidoxime ligands. Hence, our study provides a convenient means for screening suitable amidoxime monomers for future generations of poly(acrylamidoxime) adsorbents used to mine uranium from seawater.« less

  16. Acidity and hydrogen exchange dynamics of iron(II)-bound nitroxyl in aqueous solution.

    PubMed

    Gao, Yin; Toubaei, Abouzar; Kong, Xianqi; Wu, Gang

    2014-10-20

    Nitroxyl-iron(II) (HNO-Fe(II)) complexes are often unstable in aqueous solution, thus making them very difficult to study. Consequently, many fundamental chemical properties of Fe(II)-bound HNO have remained unknown. Using a comprehensive multinuclear ((1)H, (15)N, (17)O) NMR approach, the acidity of the Fe(II)-bound HNO in [Fe(CN)5(HNO)](3-) was investigated and its pK(a) value was determined to be greater than 11. Additionally, HNO undergoes rapid hydrogen exchange with water in aqueous solution and this exchange process is catalyzed by both acid and base. The hydrogen exchange dynamics for the Fe(II)-bound HNO have been characterized and the obtained benchmark values, when combined with the literature data on proteins, reveal that the rate of hydrogen exchange for the Fe(II)-bound HNO in the interior of globin proteins is reduced by a factor of 10(6). PMID:25205463

  17. Spectroscopic and photochemical properties of the lichen compound lobaric acid.

    PubMed

    Hidalgo, María Eliana; Bascuñan, Luis; Quilhot, Wanda; Fernández, Ernesto; Rubio, Cecilia

    2005-01-01

    Lichens synthesize and accumulate photoprotective compounds against possible damage induced by UV radiation in the photobiont. A biological model has been recently formulated that allows the use of lichens to evaluate changes at different UV radiation levels. The thermodynamics, photophysical and photochemical properties of lobaric acid were studied in acetonitrile, ethanol and Brij 35(3%) micelles at different pH values. Also the sun protector factor (SPF) was determined by in vitro methods. Lobaric acid was extracted from Stereoculon alpinum Laur. and characterized by means of standard procedures. Solutions were irradiated in oxygen and under nitrogen conditions with a UV medium pressure lamp. Lobaric acid absorbs at 287, 303 nm, and no fluorescence emission was observed. The maximum value of the molar extinction coefficient (5479.6 M(-1) cm(-1)) was obtained in Brij 35 at pH 12. Solubility is pH dependant and is highest in Brij 35 at pH 12 (4.45 x 10(-4) M). Photoconsumption quantum yields ranged between 10(-4) and 10(-5) in aerobic and anaerobic experimental conditions. Lobaric acid SPF was very low (0.5) compared with homosalate (4.0), (reference solar filter). Two pKa values, 5.05 (carboxylic acid group deprotonation) and 9.75 (phenolic OH deprotonation), were determined.

  18. Seasonal variations in bulk tissue, fatty acid and monosaccharide delta(13)C values of leaves from mesotrophic grassland plant communities under different grazing managements.

    PubMed

    Dungait, Jennifer A J; Docherty, Gordon; Straker, Vanessa; Evershed, Richard P

    2010-03-01

    Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide delta(13)C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific delta(13)C values could be determined. The total mean leaf bulk delta(13)C value for plants was -28.9 per thousand with a range of values spanning 7.5 per thousand. Significant interspecific variation between bulk leaf delta(13)C values was only determined in October (P=<0.001) when delta(13)C values of the leaf tissues from both sites was on average 1.5 per thousand depleted compared to during July and May. Samples from May were significantly different between fields (P=0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide delta(13)C value was 0.8 per thousand higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual delta(13)C values had a major influence on bulk delta(13)C values. An average depletion of ca. 1 per thousand in the bulk delta(13)C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1 per thousand in glucose delta(13)C values, as glucose constituted >50% total leaf monosaccharides. In October, delta(13)C values of all monosaccharides varied between species, with significant variation in delta(13)C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk delta(13)C values observed in October 1999. The fatty acids C(16:0), C(18:2) and C(18:3) were highly abundant in all plant species. Fatty acid delta(13)C values were

  19. Production of a value-added hydroxy fatty acid, 7,10-dihydroxy-8(E)-octadecenoic acid from high oleic safflower oil by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hydroxy fatty acids (HFA), originally found in small amount mainly from plant systems, are good examples of the structurally modified lipids, rendering special properties such as higher viscosity and reactivity compared to normal fatty acids. Based on these properties, HFAs possess high industrial ...

  20. D-AKAP2:PKA RII:PDZK1 ternary complex structure: insights from the nucleation of a polyvalent scaffold.

    PubMed

    Sarma, Ganapathy N; Moody, Issa S; Ilouz, Ronit; Phan, Ryan H; Sankaran, Banumathi; Hall, Randy A; Taylor, Susan S

    2015-01-01

    A-kinase anchoring proteins (AKAPs) regulate cAMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP2 (D-AKAP2/AKAP10) binds with high affinity to both RI and RII regulatory subunits of PKA and is anchored to transporters through PDZ domain proteins. Here, we describe a structure of D-AKAP2 in complex with two interacting partners and the exact mechanism by which a segment that on its own is disordered presents an α-helix to PKA and a β-strand to PDZK1. These two motifs nucleate a polyvalent scaffold and show how PKA signaling is linked to the regulation of transporters. Formation of the D-AKAP2: PKA binary complex is an important first step for high affinity interaction with PDZK1, and the structure reveals important clues toward understanding this phenomenon. In contrast to many other AKAPs, D-AKAP2 does not interact directly with the membrane protein. Instead, the interaction is facilitated by the C-terminus of D-AKAP2, which contains two binding motifs-the D-AKAP2AKB and the PDZ motif-that are joined by a short linker and only become ordered upon binding to their respective partner signaling proteins. The D-AKAP2AKB binds to the D/D domain of the R-subunit and the C-terminal PDZ motif binds to a PDZ domain (from PDZK1) that serves as a bridging protein to the transporter. This structure also provides insights into the fundamental question of why D-AKAP2 would exhibit a differential mode of binding to the two PKA isoforms. PMID:25348485

  1. Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected].

    PubMed

    Bacallao, Ketty; Monje, Paula V

    2013-01-01

    In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.

  2. Theoretical Determination of the pK a Values of Betalamic Acid Related to the Free Radical Scavenger Capacity: Comparison Between Empirical and Quantum Chemical Methods.

    PubMed

    Tutone, Marco; Lauria, Antonino; Almerico, Anna Maria

    2016-06-01

    Health benefits of dietary phytochemicals have been suggested in recent years. Among 1000s of different compounds, Betalains, which occur in vegetables of the Cariophyllalae order (cactus pear fruits and red beet), have been considered because of reducing power and potential to affect redox-modulated cellular processes. The antioxidant power of Betalains is strictly due to the dissociation rate of the acid moieties present in all the molecules of this family of phytochemicals. Experimentally, only the pK a values of betanin were determined. Recently, it was evidenced it was evidenced as the acid dissociation, at different environmental pHs, affects on its electron-donating capacity, and further on its free radical scavenging power. The identical correlation was studied on another Betalains family compound, Betalamic Acid. Experimental evidences showed that the free radical scavenging capacity of this compound drastically decreases at pH > 5, but pK a values were experimentally not measured. With the aim to justify the Betalamic Acid behavior as free radical scavenger, in this paper we tried to predict in silico the pK a values by means different approaches. Starting from the known experimental pK as of acid compounds, both phytochemicals and small organic, two empirical approaches and quantum-mechanical calculation were compared to give reliable prediction of the pK as of Betalamic Acid. Results by means these computational approaches are consistent with the experimental evidences. As shown herein, in silico, the totally dissociated species, at the experimental pH > 5 in solution, is predominant, exploiting the higher electron-donating capability (HOMO energy). Therefore, the computational estimated pK a values of Betalamic Acid resulted very reliable. PMID:26253717

  3. Reference values of whole-blood fatty acids by age and sex from European children aged 3–8 years

    PubMed Central

    Wolters, M; Schlenz, H; Foraita, R; Galli, C; Risé, P; Moreno, L A; Molnár, D; Russo, P; Veidebaum, T; Tornaritis, M; Vyncke, K; Eiben, G; Iacoviello, L; Ahrens, W

    2014-01-01

    Objectives: To establish reference values for fatty acids (FA) especially for n-3 and n-6 long-chain polyunsaturated FAs (LC PUFA) in whole-blood samples from apparently healthy 3–8-year-old European children. The whole-blood FA composition was analysed and the age- and sex-specific distribution of FA was determined. Design and subjects: Blood samples for FA analysis were taken from 2661 children of the IDEFICS (identification and prevention of dietary- and lifestyle-induced health effects in children and infants) study cohort. Children with obesity (n=454) and other diseases that are known to alter the FA composition (n=450) were excluded leaving 1653 participants in the reference population. Measurements: The FA composition of whole blood was analysed from blood drops by a rapid, validated gas chromatographic method. Results: Pearson correlation coefficients showed an age-dependent increase of C18:2n-6 and a decrease of C18:1n-9 in a subsample of normal weight boys and girls. Other significant correlations with age were weak and only seen either in boys or in girls, whereas most of the FA did not show any age dependence. For age-dependent n-3 and n-6 PUFA as well as for other FA that are correlated with age (16:0, C18:0 and C18:1n-9) percentiles analysed with the general additive model for location scale and shape are presented. A higher median in boys than in girls was observed for C20:3n-6, C20:4n-6 and C22:4n-6. Conclusions: Given the reported associations between FA status and health-related outcome, the provision of FA reference ranges may be useful for the interpretation of the FA status of children in epidemiological and clinical studies. PMID:25219413

  4. Universal reaction mechanism of boronic acids with diols in aqueous solution: kinetics and the basic concept of a conditional formation constant.

    PubMed

    Furikado, Yuki; Nagahata, Tomomi; Okamoto, Takuya; Sugaya, Tomoaki; Iwatsuki, Satoshi; Inamo, Masahiko; Takagi, Hideo D; Odani, Akira; Ishihara, Koji

    2014-10-01

    To establish a detailed reaction mechanism for the condensation between a boronic acid, RB(OH)2, and a diol, H2L, in aqueous solution, the acid dissociation constants (Ka(BL)) of boronic acid diol esters (HBLs) were determined based on the well-established concept of conditional formation constants of metal complexes. The pKa values of HBLs were 2.30, 2.77, and 2.00 for the reaction systems, 2,4-difluorophenylboronic acid and chromotropic acid, 3-nitrophenylboronic acid and alizarin red S, and phenylboronic acid and alizarin red S, respectively. A general and precise reaction mechanism of RB(OH)2 with H2L in aqueous solution, which can serve as a universal reaction mechanism for RB(OH)2 and H2L, was proposed on the basis of (a) the relative kinetic reactivities of the RB(OH)2 and its conjugate base, that is, the boronate ion, toward H2L, and (b) the determined pKa values of HBLs. The use of the conditional formation constant, K', based on the main reaction: RB(OH)2 + H2L (K1)⇌ RB(L)(OH)(-) + H3O(+) instead of the binding constant has been proposed for the general reaction of uncomplexed boronic acid species (B') with uncomplexed diol species (L') to form boronic acid diol complex species (esters, BL') in aqueous solution at pH 5-11: B' + L' (K')⇌ BL'. The proposed reaction mechanism explains perfectly the formation of boronic acid diol ester in aqueous solution.

  5. Inferring Phytoplankton, Terrestrial Plant and Bacteria Bulk δ¹³C Values from Compound Specific Analyses of Lipids and Fatty Acids

    PubMed Central

    Taipale, Sami J.; Peltomaa, Elina; Hiltunen, Minna; Jones, Roger I.; Hahn, Martin W.; Biasi, Christina; Brett, Michael T.

    2015-01-01

    Stable isotope mixing models in aquatic ecology require δ13C values for food web end members such as phytoplankton and bacteria, however it is rarely possible to measure these directly. Hence there is a critical need for improved methods for estimating the δ13C ratios of phytoplankton, bacteria and terrestrial detritus from within mixed seston. We determined the δ13C values of lipids, phospholipids and biomarker fatty acids and used these to calculate isotopic differences compared to the whole-cell δ13C values for eight phytoplankton classes, five bacterial taxa, and three types of terrestrial organic matter (two trees and one grass). The lipid content was higher amongst the phytoplankton (9.5±4.0%) than bacteria (7.3±0.8%) or terrestrial matter (3.9±1.7%). Our measurements revealed that the δ13C values of lipids followed phylogenetic classification among phytoplankton (78.2% of variance was explained by class), bacteria and terrestrial matter, and there was a strong correlation between the δ13C values of total lipids, phospholipids and individual fatty acids. Amongst the phytoplankton, the isotopic difference between biomarker fatty acids and bulk biomass averaged -10.7±1.1‰ for Chlorophyceae and Cyanophyceae, and -6.1±1.7‰ for Cryptophyceae, Chrysophyceae and Diatomophyceae. For heterotrophic bacteria and for type I and type II methane-oxidizing bacteria our results showed a -1.3±1.3‰, -8.0±4.4‰, and -3.4±1.4‰ δ13C difference, respectively, between biomarker fatty acids and bulk biomass. For terrestrial matter the isotopic difference averaged -6.6±1.2‰. Based on these results, the δ13C values of total lipids and biomarker fatty acids can be used to determine the δ13C values of bulk phytoplankton, bacteria or terrestrial matter with ± 1.4‰ uncertainty (i.e., the pooled SD of the isotopic difference for all samples). We conclude that when compound-specific stable isotope analyses become more widely available, the determination of

  6. Role of Organic Solutes in the Chemistry Of Acid-Impacted Bog Waters of the Western Czech Republic

    NASA Astrophysics Data System (ADS)

    HrušKa, Jakub; Johnson, Chris E.; KráM, Pavel

    1996-04-01

    In many regions, naturally occurring organic acid anions can effectively buffer mineral acid inputs from atmospheric deposition, moderating their effect on surface water pH. We studied the effect of chronically high inputs of acid rain on the chemistry of three brown-water streams in the western Czech Republic. The dissolved organic acids in the streams were similar in character to those of other systems in Europe and North America. The site densities (the carboxyl group content per mass of C) were similar to values reported from Fenno-Scandia, and the relationship between the apparent pKa and pH conformed to those from two North American studies. Sulfate and organic acid anions (OA-) were the dominant anions in all three streams, yet despite high dissolved organic carbon and total organic acid concentrations, OA - comprised only 21-32% of total anion charge. This pattern was due to very high sulfate concentrations and, in two of the streams, a low degree of dissociation of the organic acids, probably the results of high long-term inputs of strong acids. Stream water pH was highly correlated to sulfate concentration, but uncorrelated with OA-, suggesting that free acidity is controlled by strong mineral acids rather than organic acids. Thus future reductions in strong acid inputs should result in increased pH and a return to organic control over acid-base chemistry.

  7. 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.

  8. Conservation and divergence of the cyclic adenosine monophosphate–protein kinase A (cAMP–PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The importance of cAMP signaling in fungal development and pathogenesis has been well documented in many fungal species including several phytopathogenic Fusarium spp. Two key components of the cAMP-PKA pathway, adenylate cyclase (AC) and catalytic subunit of PKA (CPKA), have been functionally chara...

  9. Beta-Adrenergic Receptor Activation during Distinct Patterns of Stimulation Critically Modulates the PKA-Dependence of LTP in the Mouse Hippocampus

    ERIC Educational Resources Information Center

    Gelinas, Jennifer N.; Tenorio, Gustavo; Lemon, Neal; Abel, Ted; Nguyen, Peter V.

    2008-01-01

    Activation of Beta-adrenergic receptors (Beta-ARs) enhances hippocampal memory consolidation and long-term potentiation (LTP), a likely mechanism for memory storage. One signaling pathway linked to Beta-AR activation is the cAMP-PKA pathway. PKA is critical for the consolidation of hippocampal long-term memory and for the expression of some forms…

  10. Inactivation model equations and their associated parameter values obtained under static acid stress conditions cannot be used directly for predicting inactivation under dynamic conditions.

    PubMed

    Janssen, M; Verhulst, A; Valdramidis, V; Devlieghere, F; Van Impe, J F; Geeraerd, A H

    2008-11-30

    Organic acids (e.g., lactic acid, acetic acid and citric acid) are popular preservatives. In this study, the Listeria innocua inactivation is investigated under dynamic conditions of pH and undissociated lactic acid ([LaH]). A combined primary (Weibull-type) and secondary model developed for the L. innocua inactivation under static conditions [Janssen, M., Geeraerd, A.H., Cappuyns, A., Garcia-Gonzalez, L., Schockaert, G., Van Houteghem, N., Vereecken, K.M., Debevere, J., Devlieghere, F., Van Impe, J.F., 2007. Individual and combined effects of pH and lactic acid concentration on L. innocua inactivation: development of a predictive model and assessment of experimental variability. Applied and Environmental Microbiology 73(5), 1601-1611] was applied to predict the microbial inactivation under dynamic conditions. Because of its non-autonomous character, two approaches were proposed for the application of the Weibull-type model to dynamic conditions. The results quantitatively indicated that the L. innocua cell population was able to develop an induced acid stress resistance under dynamic conditions of pH and [LaH]. From a modeling point of view, it needs to be stressed that (i) inactivation model equations and associated parameter values, derived under static conditions, may not be suitable for use as such under dynamic conditions, and (ii) non-autonomous dynamic models reveal additional technical intricacies in comparison with autonomous models.

  11. The role of PKA, CaMKII, and PKC in avoidance conditioning: permissive or instructive?

    PubMed

    Shobe, Justin

    2002-05-01

    This article explores the causal and correlative relationships between kinases and learning and memory. Specifically, the contributions of three kinases-protein kinase A (PKA), calcium calmodulin-dependent kinase II (CaMKII), and protein kinase C (PKC)-are assessed during the consolidation phase of avoidance conditioning. The following sources of evidence are considered: inhibitor data, activity monitoring, and transgenic studies. An exhaustive effort is made to address several issues regarding the participation of these kinases in (a) posttraining timing and magnitude, (b) location across many brain regions, and (c) the use of multiple pharmacological agents and assays. In addition, this article attempts to integrate the behavioral data with the purported role of kinases in long-term potentiation (LTP).

  12. Phosphorylation of Complexin by PKA Regulates Activity-Dependent Spontaneous Neurotransmitter Release and Structural Synaptic Plasticity.

    PubMed

    Cho, Richard W; Buhl, Lauren K; Volfson, Dina; Tran, Adrienne; Li, Feng; Akbergenova, Yulia; Littleton, J Troy

    2015-11-18

    Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca(2+) entry. Here we show that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity.

  13. A mathematical model of pH, based on the total stoichiometric concentration of acids, bases and ampholytes dissolved in water.

    PubMed

    Mioni, Roberto; Mioni, Giuseppe

    2015-10-01

    In chemistry and in acid-base physiology, the Henderson-Hasselbalch equation plays a pivotal role in studying the behaviour of the buffer solutions. However, it seems that the general function to calculate the valence of acids, bases and ampholytes, N = f(pH), at any pH, has only been provided by Kildeberg. This equation can be applied to strong acids and bases, pluriprotic weak acids, bases and ampholytes, with an arbitrary number of acid strength constants, pKA, including water. By differentiating this function with respect to pH, we obtain the general equation for the buffer value. In addition, by integrating the titration curve, TA, proposed by Kildeberg, and calculating its Legendre transform, we obtain the Gibbs free energy of pH (or pOH)-dependent titratable acid. Starting from the law of electroneutrality and applying suitable simplifications, it is possible to calculate the pH of the buffer solutions by numerical methods, available in software packages such as Excel. The concept of buffer capacity has also been clarified by Urbansky, but, at variance with our approach, not in an organic manner. In fact, for each set of monobasic, dibasic, tribasic acids, etc., various equations are presented which independently fit each individual acid-base category. Consequently, with the increase in acid groups (pKA), the equations become more and more difficult, both in practice and in theory. Some examples are proposed to highlight the boundary that exists between acid-base physiology and the thermodynamic concepts of energy, chemical potential, amount of substance and acid resistance. PMID:26059505

  14. A mathematical model of pH, based on the total stoichiometric concentration of acids, bases and ampholytes dissolved in water.

    PubMed

    Mioni, Roberto; Mioni, Giuseppe

    2015-10-01

    In chemistry and in acid-base physiology, the Henderson-Hasselbalch equation plays a pivotal role in studying the behaviour of the buffer solutions. However, it seems that the general function to calculate the valence of acids, bases and ampholytes, N = f(pH), at any pH, has only been provided by Kildeberg. This equation can be applied to strong acids and bases, pluriprotic weak acids, bases and ampholytes, with an arbitrary number of acid strength constants, pKA, including water. By differentiating this function with respect to pH, we obtain the general equation for the buffer value. In addition, by integrating the titration curve, TA, proposed by Kildeberg, and calculating its Legendre transform, we obtain the Gibbs free energy of pH (or pOH)-dependent titratable acid. Starting from the law of electroneutrality and applying suitable simplifications, it is possible to calculate the pH of the buffer solutions by numerical methods, available in software packages such as Excel. The concept of buffer capacity has also been clarified by Urbansky, but, at variance with our approach, not in an organic manner. In fact, for each set of monobasic, dibasic, tribasic acids, etc., various equations are presented which independently fit each individual acid-base category. Consequently, with the increase in acid groups (pKA), the equations become more and more difficult, both in practice and in theory. Some examples are proposed to highlight the boundary that exists between acid-base physiology and the thermodynamic concepts of energy, chemical potential, amount of substance and acid resistance.

  15. Retinal conformation governs pKa of protonated Schiff base in rhodopsin activation.

    PubMed

    Zhu, Shengshuang; Brown, Michael F; Feller, Scott E

    2013-06-26

    We have explored the relationship between conformational energetics and the protonation state of the Schiff base in retinal, the covalently bound ligand responsible for activating the G protein-coupled receptor rhodopsin, using quantum chemical calculations. Guided by experimental structural determinations and large-scale molecular simulations on this system, we examined rotation about each bond in the retinal polyene chain, for both the protonated and deprotonated states that represent the dark and photoactivated states, respectively. Particular attention was paid to the torsional degrees of freedom that determine the shape of the molecule, and hence its interactions with the protein binding pocket. While most torsional degrees of freedom in retinal are characterized by large energetic barriers that minimize structural fluctuations under physiological temperatures, the C6-C7 dihedral defining the relative orientation of the β-ionone ring to the polyene chain has both modest barrier heights and a torsional energy surface that changes dramatically with protonation of the Schiff base. This surprising coupling between conformational degrees of freedom and protonation state is further quantified by calculations of the pKa as a function of the C6-C7 dihedral angle. Notably, pKa shifts of greater than two units arise from torsional fluctuations observed in molecular dynamics simulations of the full ligand-protein-membrane system. It follows that fluctuations in the protonation state of the Schiff base occur prior to forming the activated MII state. These new results shed light on important mechanistic aspects of retinal conformational changes that are involved in the activation of rhodopsin in the visual process.

  16. [Short-term changes of pH value and Al activity in acid soils after urea fertilization].

    PubMed

    Zeng, Qingru; Liao, Bohan; Jiang, Zhaohui; Zhou, Xihong; Tang, Can; Zhong, Ning

    2005-02-01

    Acidic soils are widely distributed in South China, and their acidity is the major environmental stress factor limiting the growth of most crops. It is well known that soil Al solubilized at low pH is a main toxic factor for plant growth. Our study with three acidic soils showed that soil pH increased quickly, while soil exchangeable Al decreased sharply with the increasing concentrations of applied urea. The time-course experiment revealed that the increase of soil pH was short-lived, with a subsequently slow drop after reached its maximum. Urea fertilization caused a drastic change of soil pH during 2-4 weeks of the experimental period. There was a negative relationship between soil pH and soil exchangeable Al. Biological toxicity test demonstrated that applying urea to acidic soils could obviously decrease the aluminum toxicity of maize in a short-term period.

  17. Peripheral involvement of PKA and PKC in subcutaneous bee venom-induced persistent nociception, mechanical hyperalgesia, and inflammation in rats.

    PubMed

    Chen, Hui-Sheng; Lei, Jing; He, Xiang; Qu, Fang; Wang, Yang; Wen, Wei-Wei; You, Hao-Jun; Arendt-Nielsen, Lars

    2008-03-01

    The roles of central protein kinases A and C (PKA and PKC) in various pain states have intensively been investigated during the past decade. The aim of the present study was to investigate the peripheral involvement of PKA and PKC in persistent nociceptive response, evoked pain behaviors, and inflammation induced by subcutaneous (s.c.) injection of bee venom (BV, 0.2mg/50 microl) in rats. The effects of intraplantar injection of H-89 (a PKA inhibitor, 5-100 microg/50 microl) and chelerythrine chloride (a PKC inhibitor, 5-100 microg/50 microl) on BV-elicited persistent nociception (nociceptive flinching reflex), mechanical hyperalgesia, and inflammation were systematically investigated. Pre-treatment with H-89 dose-dependently inhibited only BV-induced mechanical hyperalgesia, but not the persistent nociception and inflammation. In contrast, pre-treatment with chelerythrine chloride dose-dependently inhibited BV-induced sustained nociception and inflammation, but not the mechanical hyperalgesia. Topical pre-treatment of the sciatic nerve with 1% capsaicin significantly blocked the inhibitory effects of the PKC inhibitor on BV-induced inflammation, but not the persistent flinching response. These results indicate that peripheral PKA and PKC involvements in BV-induced pain behaviors differ, and capsaicin-sensitive afferents appear to participate in the pro-inflammatory role of PKC in the BV pain model. Findings from the present study also suggest that targeting specific peripheral protein kinases might prove effective in the treatment of persistent pain and inflammation.

  18. Tumor necrosis factor alpha protects heart cultures against hypoxic damage via activation of PKA and phospholamban to prevent calcium overload.

    PubMed

    El-Ani, Dalia; Philipchik, Irena; Stav, Hagit; Levi, Moran; Zerbib, Jordana; Shainberg, Asher

    2014-11-01

    This study aims to elucidate the mechanisms by which tumor necrosis factor alpha (TNFα) provides protection from hypoxic damage to neonatal rat cardiomyocyte cultures. We show that when intracellular Ca(2+) ([Ca(2+)]i) levels are elevated by extracellular Ca(2+) ([Ca(2+)]o) or by hypoxia, then TNFα decreased [Ca(2+)]i in individual cardiomyocytes. However, TNFα did not reduce [Ca(2+)]i after its increase by thapsigargin, (a SERCA2a inhibitor), indicating that TNFα attenuates Ca(2+) overload through Ca(2+) uptake by SERCA2a. TNFα did not reduce [Ca(2+)]i, following its elevation when [Ca(2+)]o levels were elevated in TNFα receptor knock-out mice. H-89, a protein kinase A (PKA) inhibitor, attenuated the protective effect of TNFα when the cardiomyoctyes were subjected to hypoxia, as determined by lactate dehydrogenase (LDH) and creatine kinase (CK) released and from the cardiomyocytes. Moreover, when the levels of [Ca(2+)]i were increased by hypoxia, H-89, but not KN93, (a calmodulin kinase II inhibitor), prevented the reduction in [Ca(2+)]i by TNFα. TNFα increased the phosphorylation of PKA in normoxic and hypoxic cardiomyoctes, indicating that the cardioprotective effect of TNFα against hypoxic damage was via PKA activation. Hypoxia decreased phosphorylated phospholamban levels; however, TNFα attenuated this decrease following hypoxia. It is suggested that TNFα activates phospholamban phosphorylation in hypoxic heart cultures via PKA to stimulate SERCA2a activity to limit Ca(2+) overload.

  19. PKA and CDK5 can phosphorylate specific serines on the intracellular domain of podoplanin (PDPN) to inhibit cell motility.

    PubMed

    Krishnan, Harini; Retzbach, Edward P; Ramirez, Maria I; Liu, Tong; Li, Hong; Miller, W Todd; Goldberg, Gary S

    2015-07-01

    Podoplanin (PDPN) is a transmembrane glycoprotein that promotes tumor cell migration, invasion, and cancer metastasis. In fact, PDPN expression is induced in many types of cancer. Thus, PDPN has emerged as a functionally relevant cancer biomarker and chemotherapeutic target. PDPN contains 2 intracellular serine residues that are conserved between species ranging from mouse to humans. Recent studies indicate that protein kinase A (PKA) can phosphorylate PDPN in order to inhibit cell migration. However, the number and identification of specific residues phosphorylated by PKA have not been defined. In addition, roles of other kinases that may phosphorylate PDPN to control cell migration have not been investigated. We report here that cyclin dependent kinase 5 (CDK5) can phosphorylate PDPN in addition to PKA. Moreover, results from this study indicate that PKA and CDK5 cooperate to phosphorylate PDPN on both intracellular serine residues to decrease cell motility. These results provide new insight into PDPN phosphorylation dynamics and the role of PDPN in cell motility. Understanding novel mechanisms of PDPN intracellular signaling could assist with designing novel targeted chemotherapeutic agents and procedures. PMID:25959509

  20. Rhes influences striatal cAMP/PKA-dependent signaling and synaptic plasticity in a gender-sensitive fashion

    PubMed Central

    Ghiglieri, Veronica; Napolitano, Francesco; Pelosi, Barbara; Schepisi, Chiara; Migliarini, Sara; Di Maio, Anna; Pendolino, Valentina; Mancini, Maria; Sciamanna, Giuseppe; Vitucci, Daniela; Maddaloni, Giacomo; Giampà, Carmela; Errico, Francesco; Nisticò, Robert; Pasqualetti, Massimo; Picconi, Barbara; Usiello, Alessandro

    2015-01-01

    Mechanisms of gender-specific synaptic plasticity in the striatum, a brain region that controls motor, cognitive and psychiatric functions, remain unclear. Here we report that Rhes, a GTPase enriched in medium spiny neurons (MSNs) of striatum, alters the striatal cAMP/PKA signaling cascade in a gender-specific manner. While Rhes knockout (KO) male mice, compared to wild-type (WT) mice, had a significant basal increase of cAMP/PKA signaling pathway, the Rhes KO females exhibited a much stronger response of this pathway, selectively under the conditions of dopamine/adenosine-related drug challenge. Corticostriatal LTP defects are exclusively found in A2AR/D2R-expressing MSNs of KO females, compared to KO males, an effect that is abolished by PKA inhibitors but not by the removal of circulating estrogens. This suggests that the synaptic alterations found in KO females could be triggered by an aberrant A2AR/cAMP/PKA activity, but not due to estrogen-mediated effect. Consistent with increased cAMP signaling, D1R-mediated motor stimulation, haloperidol-induced catalepsy and caffeine-evoked hyper-activity are robustly enhanced in Rhes KO females compared to mutant males. Thus Rhes, a thyroid hormone-target gene, plays a relevant role in gender-specific synaptic and behavioral responses. PMID:26190541

  1. Different Phases of Long-Term Memory Require Distinct Temporal Patterns of PKA Activity after Single-Trial Classical Conditioning

    ERIC Educational Resources Information Center

    Michel, Maximilian; Kemenes, Ildiko; Muller, Uli; Kemenes, Gyorgy

    2008-01-01

    The cAMP-dependent protein kinase (PKA) is known to play a critical role in both transcription-independent short-term or intermediate-term memory and transcription-dependent long-term memory (LTM). Although distinct phases of LTM already have been demonstrated in some systems, it is not known whether these phases require distinct temporal patterns…

  2. [Influence of various thermal processes and microwaves on the amino acid composition of food products: implications on their nutritional value].

    PubMed

    Gayte Sorbier, A; Audibert, M

    1978-01-01

    The cinetic study of free amino nitrogen and total soluble nitrogen of hard wheat semolina, when comparatively treated by thermal processings (convection and microwaves) shows a decrease conditioned by time and strength of heating. Aminograms show the same decrease, which is however different for each amino acid. After thermal treatment, appear ninhydrin-reactive substances, to be identified. In short, micro-wave heating seems to have the same effect as conventional heating, on free amino acids of these cereal products.

  3. (S) 2-phenyl-2-(p-tolylsulfonylamino)acetic acid. Structure, acidity and its alkali carboxylates

    NASA Astrophysics Data System (ADS)

    Duarte-Hernández, Angélica M.; Contreras, Rosalinda; Suárez-Moreno, Galdina V.; Montes-Tolentino, Pedro; Ramos-García, Iris; González, Felipe J.; Flores-Parra, Angelina

    2015-03-01

    The structure and the preferred conformers of (S) 2-phenyl-2-(p-tolylsulfonylamino)acetic acid (1) are reported. Compound 1 is a derivative of the unnatural aminoacid the (S) phenyl glycine. The X-ray diffraction analyses of the complexes of 1 with water, methanol, pyridine and its own anion are discussed. In order to add information about the acidity of the COOH and NH protons in compound 1, its pKa in DMSO and those of N-benzyl-p-tolylsulfonamide and (S) N-methylbenzyl-p-tolylsulfonamide were determined by cyclic voltammetry. Data improved the scarce information about pKa in DMSO values of sulfonamides. The products of the reactions of compound 1 with one and two equivalents of LiOH, NaOH and KOH in methanol were analyzed. Crystals of the lithium (2) and sodium (3) carboxylates and the dipotassium sulfonylamide acetate (7) were obtained, they are coordination polymers. In compound 2, the lithium is bound to four oxygen atoms with short bond lengths. The coordination of the lithium atom to two carboxylates gives an infinite ribbon by formation of fused six membered rings. In the crystal of compound 3, two pentacoordinated sodium atoms are bridged by three oxygen atoms, one from a water molecule and two from DMSO. The short distance between the sodium atoms (3.123 Å), implies a metal-metal interaction. The sodium couples are linked by two carboxylate groups, forming a planar ribbon of fused twelve membered rings. A notable discovery was a water molecule quenched in the middle of the ring, with a tetra coordinated oxygen atom in a square planar geometry. In compound 7, the carboxylate and the amide are bound to heptacoordinated potassium atoms. The 2D polymer of 7 has a sandwich structure, with the carboxylate and potassium atoms in the inner layer covered by the aromatic rings.

  4. Supercritical Fluid Extract of Spent Coffee Grounds Attenuates Melanogenesis through Downregulation of the PKA, PI3K/Akt, and MAPK Signaling Pathways

    PubMed Central

    Huang, Huey-Chun; Wei, Chien-Mei; Siao, Jen-Hung; Tsai, Tsang-Chi; Ko, Wang-Ping; Chang, Kuei-Jen; Hii, Choon-Hoon; Chang, Tsong-Min

    2016-01-01

    The mode of action of spent coffee grounds supercritical fluid CO2 extract (SFE) in melanogenesis has never been reported. In the study, the spent coffee grounds were extracted by the supercritical fluid CO2 extraction method; the chemical constituents of the SFE were investigated by gas chromatography-mass spectrometry (GC-MS). The effects of the SFE and its major fatty acid components on melanogenesis were evaluated by mushroom tyrosinase activity assay and determination of intracellular tyrosinase activity and melanin content. The expression level of melanogenesis-related proteins was analyzed by western blotting assay. The results revealed that the SFE of spent coffee grounds (1–10 mg/mL) and its major fatty acids such as linoleic acid and oleic acid (6.25–50 μM) effectively suppressed melanogenesis in the B16F10 murine melanoma cells. Furthermore, the SFE decreased the expression of melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). The SFE also decreased the protein expression levels of p-JNK, p-p38, p-ERK, and p-CREB. Our results revealed that the SFE of spent coffee grounds attenuated melanogenesis in B16F10 cells by downregulation of protein kinase A (PKA), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen-activated protein kinases (MAPK) signaling pathways, which may be due to linoleic acid and oleic acid. PMID:27375763

  5. Supercritical Fluid Extract of Spent Coffee Grounds Attenuates Melanogenesis through Downregulation of the PKA, PI3K/Akt, and MAPK Signaling Pathways.

    PubMed

    Huang, Huey-Chun; Wei, Chien-Mei; Siao, Jen-Hung; Tsai, Tsang-Chi; Ko, Wang-Ping; Chang, Kuei-Jen; Hii, Choon-Hoon; Chang, Tsong-Min

    2016-01-01

    The mode of action of spent coffee grounds supercritical fluid CO2 extract (SFE) in melanogenesis has never been reported. In the study, the spent coffee grounds were extracted by the supercritical fluid CO2 extraction method; the chemical constituents of the SFE were investigated by gas chromatography-mass spectrometry (GC-MS). The effects of the SFE and its major fatty acid components on melanogenesis were evaluated by mushroom tyrosinase activity assay and determination of intracellular tyrosinase activity and melanin content. The expression level of melanogenesis-related proteins was analyzed by western blotting assay. The results revealed that the SFE of spent coffee grounds (1-10 mg/mL) and its major fatty acids such as linoleic acid and oleic acid (6.25-50 μM) effectively suppressed melanogenesis in the B16F10 murine melanoma cells. Furthermore, the SFE decreased the expression of melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). The SFE also decreased the protein expression levels of p-JNK, p-p38, p-ERK, and p-CREB. Our results revealed that the SFE of spent coffee grounds attenuated melanogenesis in B16F10 cells by downregulation of protein kinase A (PKA), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen-activated protein kinases (MAPK) signaling pathways, which may be due to linoleic acid and oleic acid. PMID:27375763

  6. Acid-base properties of the alumina surface: influence of the titration procedures on the microcalorimetric results.

    PubMed

    Morel, Jean-Pierre; Marmier, Nicolas; Hurel, Charlotte; Morel-Desrosiers, Nicole

    2009-10-01

    The enthalpy changes associated with the protonation and deprotonation of an alumina surface have been determined on the basis of microcalorimetry experiments and acid-base potentiometric titrations at 25 degrees C. It has been shown that the results may vary significantly according to the experimental procedure. In order to do so, the potentiometric and microcalorimetric titrations have been carried out first from an acidic pH to basic pH and second from a pH near the pH(zpc) of alumina to acidic or basic pH. It has been demonstrated that the pK(a) values deduced from the potentiometric titrations are the same whatever the experimental protocol whereas the only way to obtain meaningful enthalpies of proton exchange is to carry out microcalorimetric titrations by starting around the point of zero charge.

  7. [The dynamic mitochondria-nuclear redistribution of FKBP51 during the process of adipocyte differentiation is regulated by PKA].

    PubMed

    Toneatto, Judith; Charó, Nancy L; Susperreguy, Sebastián; Piwien-Pilipuk, Graciela

    2013-01-01

    Glucocorticoids play an important role in adipogenesis via the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90-Hsp70 and a high molecular weight immunophilin FKBP51 or FKBP52. We have found that FKBP51 level of expression progressively increases, FKBP52 decreases, whereas Hsp90, Hsp70, and p23 remain unchanged when 3T3-L1 preadipocytes differentiate. Interestingly, FKBP51 translocates from mitochondria to the nucleus at the onset of adipogenesis. FKBP51 transiently concentrates in the nuclear lamina, at a time that this nuclear compartment undergoes its reorganization. FKBP51 nuclear localization is transient, after 48 h it cycles back to mitochondria. We found that the dynamic FKBP51 mitochondrial-nuclear shuttling is regulated by glucocorticoids and mainly on cAMP-PKA signaling since PKA inhibition by myristoilated-PKI, abrogated FKBP51 nuclear translocation induced by 3-isobutyl-1-methylxanthine (IBMX). It has been reported that PKA interacts with GR in a ligand dependent manner potentiating its transcriptional capacity. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced nuclear translocation of FKBP51, therefore PKA may exert a dual role in the control of GR. In summary, the presence of FKBP51 in the nucleus may be critical for GR transcriptional control, and possibly for the control of other transcription factors that are not members of the nuclear receptor family but are regulated by PKA signaling pathway, when transcription has to be strictly controlled to succeed in the acquisition of the adipocyte phenotype.

  8. [The dynamic mitochondria-nuclear redistribution of FKBP51 during the process of adipocyte differentiation is regulated by PKA].

    PubMed

    Toneatto, Judith; Charó, Nancy L; Susperreguy, Sebastián; Piwien-Pilipuk, Graciela

    2013-01-01

    Glucocorticoids play an important role in adipogenesis via the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90-Hsp70 and a high molecular weight immunophilin FKBP51 or FKBP52. We have found that FKBP51 level of expression progressively increases, FKBP52 decreases, whereas Hsp90, Hsp70, and p23 remain unchanged when 3T3-L1 preadipocytes differentiate. Interestingly, FKBP51 translocates from mitochondria to the nucleus at the onset of adipogenesis. FKBP51 transiently concentrates in the nuclear lamina, at a time that this nuclear compartment undergoes its reorganization. FKBP51 nuclear localization is transient, after 48 h it cycles back to mitochondria. We found that the dynamic FKBP51 mitochondrial-nuclear shuttling is regulated by glucocorticoids and mainly on cAMP-PKA signaling since PKA inhibition by myristoilated-PKI, abrogated FKBP51 nuclear translocation induced by 3-isobutyl-1-methylxanthine (IBMX). It has been reported that PKA interacts with GR in a ligand dependent manner potentiating its transcriptional capacity. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced nuclear translocation of FKBP51, therefore PKA may exert a dual role in the control of GR. In summary, the presence of FKBP51 in the nucleus may be critical for GR transcriptional control, and possibly for the control of other transcription factors that are not members of the nuclear receptor family but are regulated by PKA signaling pathway, when transcription has to be strictly controlled to succeed in the acquisition of the adipocyte phenotype. PMID:24152393

  9. Stimulation of oxidative phosphorylation by calcium in cardiac mitochondria is not influenced by cAMP and PKA activity.

    PubMed

    Covian, Raul; French, Stephanie; Kusnetz, Heather; Balaban, Robert S

    2014-12-01

    Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 μM free Ca2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca2 + increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 μM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 μM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca2 + stimulation of oxidative phosphorylation

  10. AMPK and PKA interaction in the regulation of survival of liver cancer cells subjected to glucose starvation

    PubMed Central

    Ferretti, Anabela C.; Tonucci, Facundo M.; Hidalgo, Florencia; Almada, Evangelina; Larocca, Maria C.; Favre, Cristián

    2016-01-01

    The signaling pathways that govern survival response in hepatic cancer cells subjected to nutritional restriction have not been clarified yet. In this study we showed that liver cancer cells undergoing glucose deprivation both arrested in G0/G1 and died mainly by apoptosis. Treatment with the AMPK activator AICAR phenocopied the effect of glucose deprivation on cell survival, whereas AMPK silencing in HepG2/C3A, HuH-7 or SK-Hep-1 cells blocked the cell cycle arrest and the increase in apoptotic death induced by glucose starvation. Both AMPK and PKA were promptly activated after glucose withdrawal. PKA signaling had a dual role during glucose starvation: whereas it elicited an early decreased in cell viability, it later improved this parameter. We detected AMPK phosphorylation (AMPKα(Ser173)) by PKA, which was increased in glucose starved cells and was associated with diminution of AMPK activation. To better explore this inhibitory effect, we constructed a hepatocarcinoma derived cell line which stably expressed an AMPK mutant lacking that PKA phosphorylation site: AMPKα1(S173C). Expression of this mutant significantly decreased viability in cells undergoing glucose starvation. Furthermore, after 36 h of glucose deprivation, the index of AMPKα1(S173C) apoptotic cells doubled the apoptotic index observed in control cells. Two main remarks arise: 1. AMPK is the central signaling kinase in the scenario of cell cycle arrest and death induced by glucose starvation in hepatic cancer cells; 2. PKA phosphorylation of Ser173 comes out as a strong control point that limits the antitumor effects of AMPK in this situation. PMID:26894973

  11. Production of a value added compound from the H-acid waste water-Bioflocculants by Klebsiella pneumoniae.

    PubMed

    Zhong, Chunying; Xu, Aihua; Wang, Buyun; Yang, Xianghui; Hong, Wentao; Yang, Baokun; Chen, Changhong; Liu, Hongtao; Zhou, Jiangang

    2014-10-01

    A novel strain (designated as ZCY-7) which could convert H-acid into bioflocculants was isolated from H-acid wastewater sludge. Conditions for bioflocculants production were optimized by response surface methodology (RSM) and determined to be inoculum size 9.65%, initial pH 7.0, and CODCr of the H-acid wastewater 520mg/L. The highest flocculating efficiency achieved for kaolin suspension was 95.1%, after 60h cultivation. The yielded bioflocculant was mainly composed of polysaccharide (82.4%) and protein (14.2%), and maintained its flocculating activity in 0.4% (w/w) kaolin suspensions over pH 2-8 and 20-80°C. Fourier transform infrared (FTIR) spectra showed that amino, amide and hydroxyl groups were present in the bioflocculant molecules. A viable alternative treatment technology of H-acid wastewater using this novel strain is suggested, which could largely reduce bioflocculants costs. In addition, flocculating mechanism investigation reveals that the bioflocculant could cause kaolin suspension instability by means of charge neutralization firstly and then promoted the aggregation of suspension particles by adsorption and bridge. It is evident from the results that H-acid wastewater could be used as a source to manufacture bioflocculants. PMID:25127749

  12. Production of a value added compound from the H-acid waste water-Bioflocculants by Klebsiella pneumoniae.

    PubMed

    Zhong, Chunying; Xu, Aihua; Wang, Buyun; Yang, Xianghui; Hong, Wentao; Yang, Baokun; Chen, Changhong; Liu, Hongtao; Zhou, Jiangang

    2014-10-01

    A novel strain (designated as ZCY-7) which could convert H-acid into bioflocculants was isolated from H-acid wastewater sludge. Conditions for bioflocculants production were optimized by response surface methodology (RSM) and determined to be inoculum size 9.65%, initial pH 7.0, and CODCr of the H-acid wastewater 520mg/L. The highest flocculating efficiency achieved for kaolin suspension was 95.1%, after 60h cultivation. The yielded bioflocculant was mainly composed of polysaccharide (82.4%) and protein (14.2%), and maintained its flocculating activity in 0.4% (w/w) kaolin suspensions over pH 2-8 and 20-80°C. Fourier transform infrared (FTIR) spectra showed that amino, amide and hydroxyl groups were present in the bioflocculant molecules. A viable alternative treatment technology of H-acid wastewater using this novel strain is suggested, which could largely reduce bioflocculants costs. In addition, flocculating mechanism investigation reveals that the bioflocculant could cause kaolin suspension instability by means of charge neutralization firstly and then promoted the aggregation of suspension particles by adsorption and bridge. It is evident from the results that H-acid wastewater could be used as a source to manufacture bioflocculants.

  13. Aqueous phase hydration and hydrate acidity of perfluoroalkyl and n:2 fluorotelomer aldehydes.

    PubMed

    Rayne, Sierra; Forest, Kaya

    2016-01-01

    The SPARC software program and comparative density functional theory (DFT) calculations were used to investigate the aqueous phase hydration equilibrium constants (Khyd) of perfluoroalkyl aldehydes (PFAlds) and n:2 fluorotelomer aldehydes (FTAlds). Both classes are degradation products of known industrial compounds and environmental contaminants such as fluorotelomer alcohols, iodides, acrylates, phosphate esters, and other derivatives, as well as hydrofluorocarbons and hydrochlorofluorocarbons. Prior studies have generally failed to consider the hydration, and subsequent potential hydrate acidity, of these compounds, resulting in incomplete and erroneous predictions as to their environmental behavior. In the current work, DFT calculations suggest that all PFAlds will be dominantly present as the hydrated form in aqueous solution. Both SPARC and DFT calculations suggest that FTAlds will not likely be substantially hydrated in aquatic systems or in vivo. PFAld hydrates are expected to have pKa values in the range of phenols (ca. 9 to 10), whereas n:2 FTAld hydrates are expected to have pKa values ca. 2 to 3 units higher (ca. 12 to 13). In order to avoid spurious modeling predictions and a fundamental misunderstanding of their fate, the molecular and/or dissociated hydrate forms of PFAlds and FTAlds need to be explicitly considered in environmental, toxicological, and waste treatment investigations. The results of the current study will facilitate a more complete examination of the environmental fate of PFAlds and FTAlds. PMID:26980678

  14. Correct pK values for dissociation constant of carbonic acid lower the reported Km values of ribulose bisphosphate carboxylase to half. Presentation of a nomograph and an equation for determining the pK values.

    PubMed

    Yokota, A; Kitaoka, S

    1985-09-30

    In the assay of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) in vitro, the concentration of CO2, the substrate of the enzyme, has been calculated from the amount of sodium bicarbonate added to the assay mixture with a dissociation constant of carbonic acid in pure water, 6.35 to 6.37. However, Rubisco is generally assayed at ionic strength of 0.1 to 0.2 M, where the dissociation constant decreases up to 6.06. The decrease of this level of the constant reduces the calculated CO2 concentration in the assay mixture to about half and accordingly the Kms of Rubisco for CO2 reported so far are not correct. The present report presents a nomograph and an equation, from which dissociation constants of carbonic acid in the presence of various concentrations of salts can be easily calculated. PMID:3931642

  15. Human baby hair amino acid natural abundance 15N-isotope values are not related to the 15N-isotope values of amino acids in mother's breast milk protein.

    PubMed

    Romek, Katarzyna M; Julien, Maxime; Frasquet-Darrieux, Marine; Tea, Illa; Antheaume, Ingrid; Hankard, Régis; Robins, Richard J

    2013-12-01

    Since exclusively breast-suckled infants obtain their nutrient only from their mother's milk, it might be anticipated that a correlation will exist between the (15)N/(14)N isotope ratios of amino acids of protein of young infants and those supplied by their mother. The work presented here aimed to determine whether amino nitrogen transfer from human milk to infant hair protein synthesized within the first month of life conserves the maternal isotopic signature or whether post-ingestion fractionation dominates the nitrogen isotope spectrum. The study was conducted at 1 month post-birth on 100 mother-infant pairs. Isotope ratios (15)N/(14)N and (13)C/(12)C were measured using isotope ratio measurement by Mass Spectrometry (irm-MS) for whole maternal milk, and infant hair and (15)N/(14)N ratios were also measured by GC-irm-MS for the N-pivaloyl-O-isopropyl esters of amino acids obtained from the hydrolysis of milk and hair proteins. The δ(15)N and δ(13)C (‰) were found to be significantly higher in infant hair than in breast milk (δ(15)N, P < 0.001; δ(13)C, P < 0.001). Furthermore, the δ(15)N (‰) of individual amino acids in infant hair was also significantly higher than that in maternal milk (P < 0.001). By calculation, the observed shift in isotope ratio was shown not to be accounted for by the amino acid composition of hair and milk proteins, indicating that it is not simply due to differences in the composition in the proteins present. Rather, it would appear that each pool-mother and infant-turns over independently, and that fractionation in infant N-metabolism even in the first month of life dominates over the nutrient N-content.

  16. DFT computation and experimental analysis of vibrational and electronic spectra of phenoxy acetic acid herbicides

    NASA Astrophysics Data System (ADS)

    Arul Dhas, D.; Hubert Joe, I.; Roy, S. D. D.; Balachandran, S.

    2013-05-01

    An absolute vibrational analysis has been attempted on the basis of experimental FTIR and NIR-FT Raman spectra with calculated vibrational wavenumbers and intensities of phenoxy acetic acids. The equilibrium geometry, bonding features and harmonic vibrational wavenumbers have been calculated with the help of B3LYP method with Dunning correlation consistent basis set aug-cc-pVTZ. The electronic structures of molecular fragments were described in terms of natural bond orbital analysis, which shows intermolecular Osbnd H⋯O and intramolecular Csbnd H⋯O hydrogen bonds. The electronic absorption spectra with different solvents have been investigated in combination with time-dependent density functional theory calculation. The pKa values of phenoxy acetic acids were compared.

  17. Activation of the cAMP-PKA signaling pathway in rat dorsal root ganglion and spinal cord contributes toward induction and maintenance of bone cancer pain.

    PubMed

    Zhu, Gui-Qin; Liu, Su; He, Duan-Duan; Liu, Yue-Peng; Song, Xue-Jun

    2014-08-01

    The objective of this study was to explore the role of cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling in the development of bone cancer pain in rats. Female Sprague-Dawley rats (N=48) were divided randomly into four groups: sham (n=8), tumor cell implantation (TCI) (n=16), TCI+saline (n=8), and TCI+PKA inhibitor (n=16). Bone cancer-induced pain behaviors - thermal hyperalgesia and mechanical allodynia - were tested at postoperative days -3, -1, 1, 3, 5, 7, 10, and 14. A PKA inhibitor, Rp-cAMPS (1 mmol/l/20 μl), was injected intrathecally on postoperative days 3, 4, and 5 (early phase) or 7, 8, and 9 postoperative days (late phase). The expression of PKA mRNA in dorsal root ganglia (DRG) was detected by reverse transcription-PCR. The concentration of cAMP and activity of PKA in DRG and spinal cord were measured by enzyme-linked immunosorbent assay. TCI treatment induced significant pain behaviors, manifested as thermal hyperalgesia and mechanical allodynia. Spinal administration of the PKA inhibitor Rp-cAMPS during the early phase and late phase significantly delayed or reversed, respectively, TCI-induced thermal hyperalgesia and mechanical allodynia. TCI treatment also led to obvious tumor growth and bone destruction. The level of PKA mRNA in the DRG, as well as the concentration of cAMP and the activity of PKA, in both the DRG and spinal cord were significantly increased after TCI treatment (P<0.01). We conclude that the inhibition of the cAMP-PKA signaling pathway may reduce bone cancer pain. PMID:24978483

  18. Correlation between precipitation and geographical location of the δ2H values of the fatty acids in milk and bulk milk powder

    NASA Astrophysics Data System (ADS)

    Ehtesham, E.; Baisden, W. T.; Keller, E. D.; Hayman, A. R.; Van Hale, R.; Frew, R. D.

    2013-06-01

    Hydrogen isotope ratios (δ2H) have become a tool for food traceability and authentication of agricultural products. The principle is that the isotopic composition of the produce is influenced by environmental and biological factors and hence exhibits a spatial differentiation of δ2H. This study investigates the variation in δ2H values of New Zealand milk, both in the bulk powder and individual fatty acids extracted from milk samples from dairy factories across New Zealand. Multivariate statistical analyses were used to test for relationships between δ2H of bulk milk powder, milk fatty acid and geographical location. Milk powder samples from different regions of New Zealand were found to exhibit patterns in isotopic composition similar to the corresponding regional precipitation associated with their origin. A model of δ2H in precipitation was developed based on measurements between 2007 and 2010 at 51 stations across New Zealand (Frew and Van Hale, 2011). The model uses multiple linear regressions to predict daily δ2H from 2 geographic and 5 rain-weighted climate variables from the 5 × 5 km New Zealand Virtual Climate Station Network (VCSN). To approximate collection radius for a drying facility the modelled values were aggregated within a 50 km radius of each dairy factory and compared to observed δ2H values of precipitation and bulk milk powder. Daily δ2H predictions for the period from August to December for the area surrounding the sample collection sites were highly correlated with the δ2H values of bulk milk powder. Therefore the δ2H value of milk fatty acids demonstrates promise as a tool for determining the provenance of milk powders and products where milk powder is an ingredient. Separation of milk powder origin to geographic sub-regions within New Zealand was achieved. Hydrogen isotope measurements could be used to complement traditional tracking systems in verifying point of origin.

  19. Ultraviolet-visible study on acid-base equilibria of aporphine alkaloids with antiplasmodial and antioxidant activities from Alseodaphne corneri and Dehaasia longipedicellata

    NASA Astrophysics Data System (ADS)

    Zahari, Azeana; Ablat, Abdulwali; Omer, Noridayu; Nafiah, Mohd Azlan; Sivasothy, Yasodha; Mohamad, Jamaludin; Khan, Mohammad Niyaz; Awang, Khalijah

    2016-02-01

    The UV-vis spectra of isocorydine 1, norisocorydine 2 and boldine 3 were studied in 2% v/v acetonitrile, at constant ionic strength (0.1 M NaCl, 35 degree Celsius). The pKa values of isocorydine 1 and norisocorydine 2 were 11.75 and 12.07, respectively. Boldine 3 gave a pKa value of 9.16 and 10.44. All of the alkaloids 1-3 were stable at physiological pH; thereby all of them will not ionize, thus permitting the basic nitrogen to be protonated and accumulated within the acidic food vacuole of Plasmodium via pH trapping. Subsequently, acidic food vacuoles that have been neutralized by alkaloids would result in enhancement of the antiplasmodial activity. The alkaloids showed antiplasmodial activity against Plasmodium falciparum and antioxidant activities; DPPH radical scavenging, metal chelating and ferric reducing power. The antioxidant properties of the alkaloids under investigation revealed that in addition to the antiplasmodial activity, the alkaloids can also prevent oxidative damage. It can be prevented by binding free heme and neutralizing the electrons produced during the Plasmodium falciparum mediated haemoglobin destruction in the host. Slightly basic properties of the aforementioned alkaloids, along with their antioxidant activities, are advantageous in improving the suppression of malaria infection that cause less damage to the host.

  20. Ultraviolet-visible study on acid-base equilibria of aporphine alkaloids with antiplasmodial and antioxidant activities from Alseodaphne corneri and Dehaasia longipedicellata

    PubMed Central

    Zahari, Azeana; Ablat, Abdulwali; Omer, Noridayu; Nafiah, Mohd Azlan; Sivasothy, Yasodha; Mohamad, Jamaludin; Khan, Mohammad Niyaz; Awang, Khalijah

    2016-01-01

    The UV-vis spectra of isocorydine 1, norisocorydine 2 and boldine 3 were studied in 2% v/v acetonitrile, at constant ionic strength (0.1 M NaCl, 35 degree Celsius). The pKa values of isocorydine 1 and norisocorydine 2 were 11.75 and 12.07, respectively. Boldine 3 gave a pKa value of 9.16 and 10.44. All of the alkaloids 1–3 were stable at physiological pH; thereby all of them will not ionize, thus permitting the basic nitrogen to be protonated and accumulated within the acidic food vacuole of Plasmodium via pH trapping. Subsequently, acidic food vacuoles that have been neutralized by alkaloids would result in enhancement of the antiplasmodial activity. The alkaloids showed antiplasmodial activity against Plasmodium falciparum and antioxidant activities; DPPH radical scavenging, metal chelating and ferric reducing power. The antioxidant properties of the alkaloids under investigation revealed that in addition to the antiplasmodial activity, the alkaloids can also prevent oxidative damage. It can be prevented by binding free heme and neutralizing the electrons produced during the Plasmodium falciparum mediated haemoglobin destruction in the host. Slightly basic properties of the aforementioned alkaloids, along with their antioxidant activities, are advantageous in improving the suppression of malaria infection that cause less damage to the host. PMID:26898753

  1. Ultraviolet-visible study on acid-base equilibria of aporphine alkaloids with antiplasmodial and antioxidant activities from Alseodaphne corneri and Dehaasia longipedicellata

    NASA Astrophysics Data System (ADS)

    Zahari, Azeana; Ablat, Abdulwali; Omer, Noridayu; Nafiah, Mohd Azlan; Sivasothy, Yasodha; Mohamad, Jamaludin; Khan, Mohammad Niyaz; Awang, Khalijah

    2016-02-01

    The UV-vis spectra of isocorydine 1, norisocorydine 2 and boldine 3 were studied in 2% v/v acetonitrile, at constant ionic strength (0.1 M NaCl, 35 degree Celsius). The pKa values of isocorydine 1 and norisocorydine 2 were 11.75 and 12.07, respectively. Boldine 3 gave a pKa value of 9.16 and 10.44. All of the alkaloids 1–3 were stable at physiological pH; thereby all of them will not ionize, thus permitting the basic nitrogen to be protonated and accumulated within the acidic food vacuole of Plasmodium via pH trapping. Subsequently, acidic food vacuoles that have been neutralized by alkaloids would result in enhancement of the antiplasmodial activity. The alkaloids showed antiplasmodial activity against Plasmodium falciparum and antioxidant activities; DPPH radical scavenging, metal chelating and ferric reducing power. The antioxidant properties of the alkaloids under investigation revealed that in addition to the antiplasmodial activity, the alkaloids can also prevent oxidative damage. It can be prevented by binding free heme and neutralizing the electrons produced during the Plasmodium falciparum mediated haemoglobin destruction in the host. Slightly basic properties of the aforementioned alkaloids, along with their antioxidant activities, are advantageous in improving the suppression of malaria infection that cause less damage to the host.

  2. cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms

    SciTech Connect

    Ghayor, Chafik; Ehrbar, Martin; Miguel, Blanca San; Graetz, Klaus W.; Weber, Franz E.

    2009-04-03

    Recent studies suggest that the elevation of intracellular cyclic adenosine monophosphate (cAMP) and the activation of the protein kinase A regulate BMP-induced osteogenesis. However, the precise mechanisms underlying the enhancing effect of cAMP on BMP2 signaling were not completely revealed. In this study we investigated the effect of elevated cAMP level and PKA activation on the BMP2-induced osteoblastic differentiation in pluripotent C2C12 cells. Alkaline phosphatase activity and its mRNA were consistently induced by BMP2 treatment. The pretreatment of C2C12 cells with Forskolin, a cAMP generating agent, dbcAMP, an analogue of cAMP, or IBMX (3-isobutyl 1-methyl xanthine), and a nonspecific inhibitor of phosphodiesterases elicited further activation of alkaline phosphatase. Furthermore, elevated intracellular cAMP level increased BMP2-induced MKP1. On the other hand, BMP2-induced Erk phosphorylation (p44/p42) and cell proliferation were suppressed in the presence of cAMP. Thus, cAMP might enhance BMP2-induced osteoblastic differentiation by a MKP1-Erk-dependent mechanism.

  3. Formulation and stability of a soap microemulsion and the apparent pK(A) herein.

    PubMed

    Marcus, Julien; Touraud, Didier; Kunz, Werner

    2013-10-01

    The influence of composition, and added salts and polyols on the stability of an oil-in-water microemulsion formulation and on the apparent pKA (apKA) of the used oleate surfactant is investigated. High temperature favours a decrease of the apKA and leads to the formation of more hydrated micelles. The apKA decreases also when the percentage of ethanol increases. Citronellol molecules do not significantly influence the apKA at concentrations between 0% and 2% w/w. By contrast, with increasing limonene concentration, the apKA increases. It was observed that anions of sodium salts destabilize the microemulsion and high temperatures are needed to recover it. By increasing the concentration of NaCl, a slight increase of the apKA is observed, which can be associated with a non-specific, electrostatic (Debye-Hückel) effect. Cations of chloride salts have different effects depending on their ability to exchange with Na(+) near the carboxylate group. Li(+), Na(+) and K(+) have apparently a salting-out effect. Tetramethylammonium chloride and choline chloride have salting-in effects until respectively 0.6 and 0.4 mol kg(-1). The associations of sorbitol or glycerol with ethanol lead to a salting-in effect and to a decrease of the apKA of Na-Oleate.

  4. pK(a) based protonation states and microspecies for protein-ligand docking.

    PubMed

    ten Brink, Tim; Exner, Thomas E

    2010-11-01

    In this paper we present our reworked approach to generate ligand protonation states with our structure preparation tool SPORES (Structure PrOtonation and REcognition System). SPORES can be used for the preprocessing of proteins and protein-ligand complexes as e.g. taken from the Protein Data Bank as well as for the setup of 3D ligand databases. It automatically assigns atom and bond types, generates different protonation, tautomeric states as well as different stereoisomers. In the revised version, pKa calculations with the ChemAxon software MARVIN are used either to determine the likeliness of a combinatorial generated protonation state or to determine the titrable atoms used in the combinatorial approach. Additionally, the MARVIN software is used to predict microspecies distributions of ligand molecules. Docking studies were performed with our recently introduced program PLANTS (Protein-Ligand ANT System) on all protomers resulting from the three different selection methods for the well established CCDC/ASTEX clean data set demonstrating the usefulness of especially the latter approach.

  5. PKA regulatory IIα subunit is essential for PGD2-mediated resolution of inflammation.

    PubMed

    Kong, Deping; Shen, Yujun; Liu, Guizhu; Zuo, Shengkai; Ji, Yong; Lu, Ankang; Nakamura, Masataka; Lazarus, Michael; Stratakis, Constantine A; Breyer, Richard M; Yu, Ying

    2016-09-19

    The kinetic participation of macrophages is critical for inflammatory resolution and recovery from myocardial infarction (MI), particularly with respect to the transition from the M1 to the M2 phenotype; however, the underlying mechanisms are poorly understood. In this study, we found that the deletion of prostaglandin (PG) D2 receptor subtype 1 (DP1) in macrophages retarded M2 polarization, antiinflammatory cytokine production, and resolution in different inflammatory models, including the MI model. DP1 deletion up-regulated proinflammatory genes expression via JAK2/STAT1 signaling in macrophages, whereas its activation facilitated binding of the separated PKA regulatory IIα subunit (PRKAR2A) to the transmembrane domain of IFN-γ receptor, suppressed JAK2-STAT1 axis-mediated M1 polarization, and promoted resolution. PRKAR2A deficiency attenuated DP1 activation-mediated M2 polarization and resolution of inflammation. Collectively, PGD2-DP1 axis-induced M2 polarization facilitates resolution of inflammation through the PRKAR2A-mediated suppression of JAK2/STAT1 signaling. These observations indicate that macrophage DP1 activation represents a promising strategy in the management of inflammation-associated diseases, including post-MI healing. PMID:27621415

  6. pK(a) based protonation states and microspecies for protein-ligand docking.

    PubMed

    ten Brink, Tim; Exner, Thomas E

    2010-11-01

    In this paper we present our reworked approach to generate ligand protonation states with our structure preparation tool SPORES (Structure PrOtonation and REcognition System). SPORES can be used for the preprocessing of proteins and protein-ligand complexes as e.g. taken from the Protein Data Bank as well as for the setup of 3D ligand databases. It automatically assigns atom and bond types, generates different protonation, tautomeric states as well as different stereoisomers. In the revised version, pKa calculations with the ChemAxon software MARVIN are used either to determine the likeliness of a combinatorial generated protonation state or to determine the titrable atoms used in the combinatorial approach. Additionally, the MARVIN software is used to predict microspecies distributions of ligand molecules. Docking studies were performed with our recently introduced program PLANTS (Protein-Ligand ANT System) on all protomers resulting from the three different selection methods for the well established CCDC/ASTEX clean data set demonstrating the usefulness of especially the latter approach. PMID:20882397

  7. Dynamic monitoring of plasma amino acids and carnitine during chemotherapy of patients with alimentary canal malignancies and its clinical value

    PubMed Central

    Wang, Xiaoyu; Wang, Jiaqi; Wang, Zhenghua; Wang, Qingjun; Li, Hua

    2015-01-01

    Objective The aim of this study was to observe the plasma amino acid and carnitine characteristics in patients with metastatic gastrointestinal malignancies during chemotherapy and to identify markers for the early diagnosis and evaluation of adverse reactions and prognosis of the digestive tract malignant tumor patients. Methods Blood samples of 30 patients with metastatic gastrointestinal malignancies were collected at four time points: before chemotherapy, the first day after chemotherapy (+1 day), bone marrow depression period (+14 days), and hematopoietic recovery period (+21 days). The plasma amino acids and carnitine from those 30 patients were determined by high-performance liquid chromatography–tandem mass spectrometry method. Simultaneously, the levels of 21 amino acids were detected in 30 healthy individuals, who were considered as control. Biochemical indexes were also detected at four time points, adverse reactions were recorded during the chemotherapy process, and patients were followed up for 1 year to observe time to progression (TTP) and progression-free survival (PFS). Results Compared to healthy people in the control group, patients with malignancies showed significantly increased levels of plasma amino acids such as Arg, Asp, Cit, Gly, Orn, Tyr, Val, and carnitine (such as C2). The levels of compounds such as C3, Asn, Leu, Lys, Pip, Pro, C0, C5:1 decreased significantly before chemotherapy. The levels of Cit, Cys, Lys, Pro, Tyr, Val, C0, and C2 decreased significantly on the second day of chemotherapy (+1 day), whereas the level of C3 increased significantly. During myelosuppression (+14 days), the levels of Asp, Cit, Met, and Orn were observed to still decrease significantly, whereas the level of Val appeared to increase significantly. The levels of Asp, Glu, and Met were clearly different among patients with gastric carcinoma, rectal cancer, and colon cancer. Compared to the control group, aspartate amino transferase and alanine

  8. Intrinsic acid-base properties of a hexa-2'-deoxynucleoside pentaphosphate, d(ApGpGpCpCpT): neighboring effects and isomeric equilibria.

    PubMed

    Domínguez-Martín, Alicia; Johannsen, Silke; Sigel, Astrid; Operschall, Bert P; Song, Bin; Sigel, Helmut; Okruszek, Andrzej; González-Pérez, Josefa María; Niclós-Gutiérrez, Juan; Sigel, Roland K O

    2013-06-17

    The intrinsic acid-base properties of the hexa-2'-deoxynucleoside pentaphosphate, d(ApGpGpCpCpT) [=(A1∙G2∙G3∙C4∙C5∙T6)=(HNPP)⁵⁻] have been determined by ¹H NMR shift experiments. The pKa values of the individual sites of the adenosine (A), guanosine (G), cytidine (C), and thymidine (T) residues were measured in water under single-strand conditions (i.e., 10% D₂O, 47 °C, I=0.1 M, NaClO₄). These results quantify the release of H⁺ from the two (N7)H⁺ (G∙G), the two (N3)H⁺ (C∙C), and the (N1)H⁺ (A) units, as well as from the two (N1)H (G∙G) and the (N3)H (T) sites. Based on measurements with 2'-deoxynucleosides at 25 °C and 47 °C, they were transferred to pKa values valid in water at 25 °C and I=0.1 M. Intramolecular stacks between the nucleobases A1 and G2 as well as most likely also between G2 and G3 are formed. For HNPP three pKa clusters occur, that is those encompassing the pKa values of 2.44, 2.97, and 3.71 of G2(N7)H⁺, G3(N7)H⁺, and A1(N1)H⁺, respectively, with overlapping buffer regions. The tautomer populations were estimated, giving for the release of a single proton from five-fold protonated H₅(HNPP)(±) , the tautomers (G2)N7, (G3)N7, and (A1)N1 with formation degrees of about 74, 22, and 4%, respectively. Tautomer distributions reveal pathways for proton-donating as well as for proton-accepting reactions both being expected to be fast and to occur practically at no "cost". The eight pKa values for H₅(HNPP)(±) are compared with data for nucleosides and nucleotides, revealing that the nucleoside residues are in part affected very differently by their neighbors. In addition, the intrinsic acidity constants for the RNA derivative r(A1∙G2∙G3∙C4∙C5∙U6), where U=uridine, were calculated. Finally, the effect of metal ions on the pKa values of nucleobase sites is briefly discussed because in this way deprotonation reactions can easily be shifted to the physiological pH range.

  9. PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation.

    PubMed

    Firulli, Beth A; Howard, Marthe J; McDaid, Jennifer R; McIlreavey, Leanne; Dionne, Karen M; Centonze, Victoria E; Cserjesi, Peter; Virshup, David M; Firulli, Anthony B

    2003-11-01

    The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.

  10. Silicic acid competes for dimethylarsinic acid (DMA) immobilization by the iron hydroxide plaque mineral goethite.

    PubMed

    Kersten, Michael; Daus, Birgit

    2015-03-01

    A surface complexation modeling approach was used to extend the knowledge about processes that affect the availability of dimethylarsinic acid (DMA) in the soil rhizosphere in presence of a strong sorbent, e.g., Fe plaques on rice roots. Published spectroscopic and molecular modeling information suggest for the organoarsenical agent to form bidentate-binuclear inner-sphere surface complexes with Fe hydroxides similar to the inorganic As oxyanions. However, since also the ubiquitous silicic acid oxyanion form the same bidentate binuclear surface complexes, our hypothesis was that it may have an effect on the adsorption of DMA by Fe hydroxides in soil. Our experimental batch equilibrium data show that DMA is strongly adsorbed in the acidic pH range, with a steep adsorption edge in the circumneutral pH region between the DMA acidity constant (pKa=6.3) and the point of zero charge value of the goethite adsorbent (pHpzc=8.6). A 1-pK CD-MUSIC surface complexation model was chosen to fit the experimental adsorption vs. pH data. The same was done for silicic acid batch equilibrium data with our goethite adsorbent. Both model parameters for individual DMA and silicic acid adsorption were then merged into one CD-MUSIC model to predict the binary DMA+Si adsorption behavior. Silicic acid (500 μM) was thus predicted by the model to strongly compete for DMA with up to 60% mobilization of the latter at a pH6. This model result could be verified subsequently by experimental batch equilibrium data with zero adjustable parameters. The thus quantified antagonistic relation between DMA and silicic acid is discussed as one of factors to explain the increase of the DMA proportion in rice grains as observed upon silica fertilization of rice fields. PMID:25478657

  11. Determining Partition Coefficient (Log P), Distribution Coefficient (Log D) and Ionization Constant (pKa) in Early Drug Discovery.

    PubMed

    Bharate, Sonali S; Kumar, Vikas; Vishwakarma, Ram A

    2016-01-01

    An early prediction of physicochemical properties is highly desirable during drug discovery to find out a viable lead candidate. Although there are several methods available to determine partition coefficient (log P), distribution coefficient (log D) and ionization constant (pKa), none of them involves simple and fixed, miniaturized protocols for diverse set of compounds. Therefore, it is necessary to establish simple, uniform and medium-throughput protocols requiring small sample quantities for the determination of these physicochemical properties. Log P and log D were determined by shake flask method, wherein, the compound was partitioned between presaturated noctanol and water phase (water/PBS pH 7.4) and the concentration of compound in each phase was determined by HPLC. The pKa determination made use of UV spectrophotometric analysis in a 96-well microtiter plate containing a series of aqueous buffers ranging from pH 1.0 to 13.0. The medium-throughput miniaturized protocols described herein, for determination of log P, log D and pKa, are straightforward to set up and require very small quantities of sample (< 5 mg for all three properties). All established protocols were validated using diverse set of compounds.

  12. PKA antagonizes CLASP-dependent microtubule stabilization to re-localize Pom1 and buffer cell size upon glucose limitation

    PubMed Central

    Kelkar, Manasi; Martin, Sophie G.

    2015-01-01

    Cells couple growth with division and regulate size in response to nutrient availability. In rod-shaped fission yeast, cell-size control occurs at mitotic commitment. An important regulator is the DYRK-family kinase Pom1, which forms gradients from cell poles and inhibits the mitotic activator Cdr2, itself localized at the medial cortex. Where and when Pom1 modulates Cdr2 activity is unclear as Pom1 medial cortical levels remain constant during cell elongation. Here we show that Pom1 re-localizes to cell sides upon environmental glucose limitation, where it strongly delays mitosis. This re-localization is caused by severe microtubule destabilization upon glucose starvation, with microtubules undergoing catastrophe and depositing the Pom1 gradient nucleator Tea4 at cell sides. Microtubule destabilization requires PKA/Pka1 activity, which negatively regulates the microtubule rescue factor CLASP/Cls1/Peg1, reducing CLASP's ability to stabilize microtubules. Thus, PKA signalling tunes CLASP's activity to promote Pom1 cell side localization and buffer cell size upon glucose starvation. PMID:26443240

  13. Tor1, Sch9 and PKA downregulation in quiescence rely on Mtl1 to preserve mitochondrial integrity and cell survival.

    PubMed

    Sundaram, Venkatraghavan; Petkova, Mima I; Pujol-Carrion, Nuria; Boada, Jordi; de la Torre-Ruiz, Maria Angeles

    2015-07-01

    Here we show that Mtl1, member of the cell wall integrity pathway of Saccharomyces cerevisiae, plays a positive role in chronological life span (CLS). The absence of Mtl1 shortens CLS and causes impairment in the mitochondrial function. This is reflected in a descent in oxygen consumption during the postdiauxic state, an increase in the uncoupled respiration and mitochondrial membrane potential and also a descent in aconitase activity. We demonstrate that all these effects are a consequence of signalling defects suppressed by TOR1 (target of rapamycin) and SCH9 deletion and less efficiently by Protein kinase A (PKA) inactivation. Mtl1 also plays a role in the regulation of both Bcy1 stability and phosphorylation, mainly in response to glucose depletion. In postdiauxic phase and in conditions of glucose depletion, Mtl1 negatively regulates TOR1 function leading to Sch9 inactivation and Bcy1 phosphorylation converging in PKA inhibition. Slt2/Mpk1 kinase partially contributes to Bcy1 phosphorylation, although additional targets are not excluded. Mtl1 links mitochondrial dysfunction with TOR and PKA pathways in quiescence, glucose being the main signalling molecule.

  14. Determining Partition Coefficient (Log P), Distribution Coefficient (Log D) and Ionization Constant (pKa) in Early Drug Discovery.

    PubMed

    Bharate, Sonali S; Kumar, Vikas; Vishwakarma, Ram A

    2016-01-01

    An early prediction of physicochemical properties is highly desirable during drug discovery to find out a viable lead candidate. Although there are several methods available to determine partition coefficient (log P), distribution coefficient (log D) and ionization constant (pKa), none of them involves simple and fixed, miniaturized protocols for diverse set of compounds. Therefore, it is necessary to establish simple, uniform and medium-throughput protocols requiring small sample quantities for the determination of these physicochemical properties. Log P and log D were determined by shake flask method, wherein, the compound was partitioned between presaturated noctanol and water phase (water/PBS pH 7.4) and the concentration of compound in each phase was determined by HPLC. The pKa determination made use of UV spectrophotometric analysis in a 96-well microtiter plate containing a series of aqueous buffers ranging from pH 1.0 to 13.0. The medium-throughput miniaturized protocols described herein, for determination of log P, log D and pKa, are straightforward to set up and require very small quantities of sample (< 5 mg for all three properties). All established protocols were validated using diverse set of compounds. PMID:27137915

  15. Constitutive activity of H3 autoreceptors modulates histamine synthesis in rat brain through the cAMP/PKA pathway.

    PubMed

    Moreno-Delgado, David; Torrent, Anna; Gómez-Ramírez, Jordi; de Esch, Iwan; Blanco, Isaac; Ortiz, Jordi

    2006-09-01

    We previously described that agonist-activated histamine H3 autoreceptors inhibit the stimulation of histamine synthesis mediated by calcium/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA respectively) in histaminergic nerve endings. In the absence of an agonist H3 receptors show partial constitutive activity, so we hypothesized that suppression of constitutive activity by an inverse agonist could stimulate these transduction pathways. We show here that the H3 inverse agonist thioperamide increases histamine synthesis in rat brain cortical slices independently from the amounts of extracellular histamine. Thioperamide effects were mimicked by the inverse agonists clobenpropit and A-331440, but not by the neutral antagonist VUF-5681. In contrast, coincubation with VUF-5681 suppressed thioperamide effects. The effects of thioperamide were completely blocked by the PKA inhibitor peptide myristoyl-PKI14-22, a peptide that did not block depolarization stimulation of histamine synthesis. In addition, thioperamide effects required depolarization and were impaired by blockade of N-type calcium channels (mediating depolarization), but not by CaMKII inhibition. These results indicate that constitutive activity of H3 receptors in rat brain cortex inhibits the adenylate cyclase/PKA pathway, and perhaps also the opening of N-type voltage sensitive calcium channels, but apparently not CaMKII.

  16. Regulation of glutamate receptor internalization by the spine cytoskeleton is mediated by its PKA-dependent association with CPG2.

    PubMed

    Loebrich, Sven; Djukic, Biljana; Tong, Zachary J; Cottrell, Jeffrey R; Turrigiano, Gina G; Nedivi, Elly

    2013-11-19

    A key neuronal mechanism for adjusting excitatory synaptic strength is clathrin-mediated endocytosis of postsynaptic glutamate receptors (GluRs). The actin cytoskeleton is critical for clathrin-mediated endocytosis, yet we lack a mechanistic understanding of its interaction with the endocytic process and how it may be regulated. Here we show that F-actin in dendritic spines physically binds the synaptic nuclear envelope 1 gene product candidate plasticity gene 2 (CPG2) in a PKA-dependent manner, and that this association is required for synaptic GluR internalization. Mutating two PKA sites on CPG2 disrupts its cytoskeletal association, attenuating GluR endocytosis and affecting the efficacy of synaptic transmission in vivo. These results identify CPG2 as an F-actin binding partner that functionally mediates interaction of the spine cytoskeleton with postsynaptic endocytosis. Further, the regulation of CPG2/F-actin association by PKA provides a gateway for cellular control of synaptic receptor internalization through second messenger signaling pathways. Recent identification of human synaptic nuclear envelope 1 as a risk locus for bipolar disorder suggests that CPG2 could play a role in synaptic dysfunction underlying neuropsychiatric disease. PMID:24191017

  17. Regulation of glutamate receptor internalization by the spine cytoskeleton is mediated by its PKA-dependent association with CPG2

    PubMed Central

    Loebrich, Sven; Djukic, Biljana; Tong, Zachary J.; Cottrell, Jeffrey R.; Turrigiano, Gina G.; Nedivi, Elly

    2013-01-01

    A key neuronal mechanism for adjusting excitatory synaptic strength is clathrin-mediated endocytosis of postsynaptic glutamate receptors (GluRs). The actin cytoskeleton is critical for clathrin-mediated endocytosis, yet we lack a mechanistic understanding of its interaction with the endocytic process and how it may be regulated. Here we show that F-actin in dendritic spines physically binds the synaptic nuclear envelope 1 gene product candidate plasticity gene 2 (CPG2) in a PKA-dependent manner, and that this association is required for synaptic GluR internalization. Mutating two PKA sites on CPG2 disrupts its cytoskeletal association, attenuating GluR endocytosis and affecting the efficacy of synaptic transmission in vivo. These results identify CPG2 as an F-actin binding partner that functionally mediates interaction of the spine cytoskeleton with postsynaptic endocytosis. Further, the regulation of CPG2/F-actin association by PKA provides a gateway for cellular control of synaptic receptor internalization through second messenger signaling pathways. Recent identification of human synaptic nuclear envelope 1 as a risk locus for bipolar disorder suggests that CPG2 could play a role in synaptic dysfunction underlying neuropsychiatric disease. PMID:24191017

  18. Spinal Activation of the cAMP-PKA Pathway Induces Respiratory Motor Recovery Following High Cervical Spinal Cord Injury

    PubMed Central

    Kajana, S.; Goshgarian, H.G.

    2008-01-01

    The present study investigated the involvement of the adenosine 3’5’-cyclic monophosphate-dependent protein kinase A (cAMP-PKA) pathway in the activation of the crossed phrenic pathways after left C2 spinal cord hemisection. Experiments were conducted on left C2 spinal cord hemisected, anesthetized, vagotomized, pancuronium paralyzed, and artificially ventilated male Sprague-Dawley rats. One week post-injury, the ipsilateral phrenic nerve exhibited no respiratory-related activity indicating a functionally complete hemisection. Intrathecal spinal cord administration of the cAMP analog, 8-Br-cAMP at the level of the phrenic nucleus resulted in an enhancement of contralateral phrenic nerve output and a restoration of respiratory-related activity in the phrenic nerve ipsilateral to the hemisection. Furthermore, pretreatment with Rp-8-Br-cAMP, a PKA inhibitor, abolished the effects of 8-Br-cAMP. These results suggest that PKA activation is necessary for the cAMP-mediated respiratory recovery following high cervical spinal cord injury and that activation of intracellular signaling cascades may represent an important strategy for improving respiratory function after spinal cord injury. PMID:18656458

  19. Arsenic may be involved in fluoride-induced bone toxicity through PTH/PKA/AP1 signaling pathway.

    PubMed

    Zeng, Qi-bing; Xu, Yu-yan; Yu, Xian; Yang, Jun; Hong, Feng; Zhang, Ai-hua

    2014-01-01

    Chronic exposure to combined fluoride and arsenic continues to be a major public health problem worldwide, affecting thousands of people. In recent years, more and more researchers began to focus on the interaction between the fluorine and the arsenic. In this study, the selected investigation site was located in China. The study group was selected from people living in fluoride-arsenic polluted areas due to burning coal. The total number of participants was 196; including the fluoride-arsenic anomaly group (130) and the fluoride-arsenic normal group (63). By observing the changes in gene and protein expression of PTH/PKA/AP1 signaling pathway, the results show that fluoride can increase the expression levels of PTH, PKA, and AP1, but arsenic can only affect the expression of AP1; fluoride and arsenic have an interaction on the expression of AP1. Further study found that fluoride and arsenic can affect the mRNA expression level of c-fos gene (AP1 family members), and have an interaction on the expression of c-fos, but not c-jun. The results indicate that PTH/PKA/AP1 signaling pathway may play an important role in bone toxicity of fluoride. Arsenic can affect the expression of c-fos, thereby affecting the expression of transcription factor AP1, indirectly involved in fluoride-induced bone toxicity.

  20. Intrinsic, Pro-Apoptotic Effects of IGFBP-3 on Breast Cancer Cells are Reversible: Involvement of PKA, Rho, and Ceramide

    PubMed Central

    Perks, Claire M.; Burrows, Carla; Holly, Jeff M. P.

    2011-01-01

    We established previously that IGFBP-3 could exert positive or negative effects on cell function depending upon the extracellular matrix composition and by interacting with integrin signaling. To elicit its pro-apoptotic effects IGFBP-3 bound to caveolin-1 and the beta 1 integrin receptor and increased their association culminating in MAPK activation. Disruption of these complexes or blocking the beta 1 integrin receptor reversed these intrinsic actions of IGFBP-3. In this study we have examined the signaling pathway between integrin receptor binding and MAPK activation that mediates the intrinsic, pro-apoptotic actions of IGFBP-3. We found on inhibiting protein kinase A (PKA), Rho associated kinase (ROCK), and ceramide, the accentuating effects of IGFBP-3 on apoptotic triggers were reversed, such that IGFBP-3 then conferred cell survival. We established that IGFBP-3 activated Rho, the upstream regulator of ROCK and that beta1 integrin and PKA were upstream of Rho activation, whereas the involvement of ceramide was downstream. The beta 1 integrin, PKA, Rho, and ceramide were all upstream of MAPK activation. These data highlight key components involved in the pro-apoptotic effects of IGFBP-3 and that inhibiting them leads to a reversal in the action of IGFBP-3. PMID:22654794

  1. "One-step production of biodiesel from Jatropha oil with high-acid value in ionic liquids" [Bioresour. Technol. 102 (11) (2011)].

    PubMed

    Guo, Feng; Fang, Zhen; Tian, Xiao-fei; Long, Yun-Duo; Jiang, Li-Qun

    2013-07-01

    Catalytic conversion of un-pretreated Jatropha oil with high-acid value (13.8 mg KOH/g) to biodiesel was studied in ionic liquids (ILs) with metal chlorides. Several commercial ILs were used to catalyze the esterification of oleic acid. It was found that 1-butyl-3-methylimidazolium tosylate {[BMIm][TS]} had high catalytic activity with 93% esterification rate for oleic acid at 140 °C but only 63.7% Jatropha biodiesel yield at 200 °C. When ZnCl2 was added to [BMIm][TS], a maximum Jatropha biodiesel yield of 92.5% was achieved at 180 °C. Addition of metal ions supplied Lewis acidic sites in ILs promoted both esterification and transestrification reactions. It was also found that the transition metal ions performed higher catalytic activity in transestrification than the ions of Group A. Mixture of [BMIm][TS] and ZnCl2 was easily separated from products for reuse to avoid producing pollutants. PMID:23908993

  2. Quantitative structure-permeability relationships at various pH values for acidic and basic drugs and drug-like compounds.

    PubMed

    Oja, M; Maran, U

    2015-01-01

    Absorption in gastrointestinal tract compartments varies and is largely influenced by pH. Therefore, considering pH in studies and analyses of membrane permeability provides an opportunity to gain a better understanding of the behaviour of compounds and to obtain good permeability estimates for prediction purposes. This study concentrates on relationships between the chemical structure and membrane permeability of acidic and basic drugs and drug-like compounds. The membrane permeability of 36 acidic and 61 basic compounds was measured using the parallel artificial membrane permeability assay (PAMPA) at pH 3, 5, 7.4 and 9. Descriptive and/or predictive single-parameter quantitative structure-permeability relationships were derived for all pH values. For acidic compounds, membrane permeability is mainly influenced by hydrogen bond donor properties, as revealed by models with r(2) > 0.8 for pH 3 and pH 5. For basic compounds, the best (r(2) > 0.7) structure-permeability relationships are obtained with the octanol-water distribution coefficient for pH 7.4 and pH 9, indicating the importance of partition properties. In addition to the validation set, the prediction quality of the developed models was tested with folic acid and astemizole, showing good matches between experimental and calculated membrane permeabilities at key pHs. Selected QSAR models are available at the QsarDB repository ( http://dx.doi.org/10.15152/QDB.166 ). PMID:26383235

  3. Influence of nitrogen source and pH value on undesired poly(γ-glutamic acid) formation of a protease producing Bacillus licheniformis strain.

    PubMed

    Meissner, Lena; Kauffmann, Kira; Wengeler, Timo; Mitsunaga, Hitoshi; Fukusaki, Eiichiro; Büchs, Jochen

    2015-09-01

    Bacillus spp. are used for the production of industrial enzymes but are also known to be capable of producing biopolymers such as poly(γ-glutamic acid). Biopolymers increase the viscosity of the fermentation broth, thereby impairing mixing, gas/liquid mass and heat transfer in any bioreactor system. Undesired biopolymer formation has a significant impact on the fermentation and downstream processing performance. This study shows how undesirable poly(γ-glutamic acid) formation of an industrial protease producing Bacillus licheniformis strain was prevented by switching the nitrogen source from ammonium to nitrate. The viscosity was reduced from 32 to 2.5 mPa s. A constant or changing pH value did not influence the poly(γ-glutamic acid) production. Protease production was not affected: protease activities of 38 and 46 U mL(-1) were obtained for ammonium and nitrate, respectively. With the presented results, protease production with industrial Bacillus strains is now possible without the negative impact on fermentation and downstream processing by undesired poly(γ-glutamic acid) formation.

  4. Assessing the Indirect Photochemical Transformation of Dissolved Combined Amino Acids through the Use of Systematically Designed Histidine-Containing Oligopeptides.

    PubMed

    Chu, Chiheng; Lundeen, Rachel A; Sander, Michael; McNeill, Kristopher

    2015-11-01

    Photooxidation is an important abiotic transformation pathway for amino acids (AAs) in sunlit waters. Although dissolved free AAs are well studied, the photooxidation of dissolved combined AAs (DCAAs) remains poorly investigated. This study is a systematic investigation of the effect of neighboring photostable AA residues (i.e., aliphatic, cationic, anionic, or aromatic residues) on the environmental indirect photochemical transformation of histidine (His) in His-containing oligopeptides. The pKa values of His residues in the studied oligopeptides were found to be between 4.3 and 8.1. Accordingly, the phototransformation rate constants of the His-containing oligopeptides were highly pH-dependent in an environmentally relevant pH range with higher reactivity for neutral His than for the protonated species. The photostable AA residues significantly modulated the photoreactivity of oligopeptides either through altering the accessibility of His to photochemically produced oxidants or through shifting the pKa values of His residues. In addition, the influence of neighboring photostable AA residues on the sorption-enhanced phototransformation of oligopeptides in solutions containing chromophoric dissolved organic matter (CDOM) was assessed. The constituent photostable AA residues promoted sorption of His-containing oligopeptides to CDOM macromolecules through electrostatic attraction, hydrophobic effects, and/or low-barrier hydrogen bonds, and subsequently increased the apparent phototransformation rate constants by up to 2 orders of magnitude.

  5. A computational study of ultrafast acid dissociation and acid-base neutralization reactions. I. The model

    NASA Astrophysics Data System (ADS)

    Maurer, Patrick; Thomas, Vibin; Rivard, Ugo; Iftimie, Radu

    2010-07-01

    Ultrafast, time-resolved investigations of acid-base neutralization reactions have recently been performed using systems containing the photoacid 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) and various Brønsted bases. Two conflicting neutralization mechanisms have been formulated by Mohammed et al. [Science 310, 83 (2005)] and Siwick et al. [J. Am. Chem. Soc. 129, 13412 (2007)] for the same acid-base system. Herein an ab initio molecular dynamics based computational model is formulated, which is able to investigate the validity of the proposed mechanisms in the general context of ground-state acid-base neutralization reactions. Our approach consists of using 2,4,6-tricyanophenol (exp. pKa≅1) as a model for excited-state HPTS∗ (pKa≅1.4) and carboxylate ions for the accepting base. We employ our recently proposed dipole-field/quantum mechanics (QM) treatment [P. Maurer and R. Iftimie, J. Chem. Phys. 132, 074112 (2010)] of the proton donor and acceptor molecules. This approach allows one to tune the free energy of neutralization to any desired value as well as model initial nonequilibrium hydration effects caused by a sudden increase in acidity, making it possible to achieve a more realistic comparison with experimental data than could be obtained via a full-QM treatment of the entire system. It is demonstrated that the dipole-field/QM model reproduces correctly key properties of the 2,4,6-tricyanophenol acid molecule including gas-phase proton dissociation energies and dipole moments, and condensed-phase hydration structure and pKa values.

  6. Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production.

    PubMed

    Mooibroek, Hans; Oosterhuis, Nico; Giuseppin, Marco; Toonen, Marcel; Franssen, Henk; Scott, Elinor; Sanders, Johan; Steinbüchel, Alexander

    2007-11-01

    Major transitions can be expected within the next few decades aiming at the reduction of pollution and global warming and at energy saving measures. For these purposes, new sustainable biorefinery concepts will be needed that will replace the traditional mineral oil-based synthesis of specialty and bulk chemicals. An important group of these chemicals are those that comprise N-functionalities. Many plant components contained in biomass rest or waste stream fractions contain these N-functionalities in proteins and free amino acids that can be used as starting materials for the synthesis of biopolymers and chemicals. This paper describes the economic and technological feasibility for cyanophycin production by fermentation of the potato waste stream Protamylassetrade mark or directly in plants and its subsequent conversion to a number of N-containing bulk chemicals.

  7. Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production

    PubMed Central

    Oosterhuis, Nico; Giuseppin, Marco; Toonen, Marcel; Franssen, Henk; Scott, Elinor; Sanders, Johan; Steinbüchel, Alexander

    2007-01-01

    Major transitions can be expected within the next few decades aiming at the reduction of pollution and global warming and at energy saving measures. For these purposes, new sustainable biorefinery concepts will be needed that will replace the traditional mineral oil-based synthesis of specialty and bulk chemicals. An important group of these chemicals are those that comprise N-functionalities. Many plant components contained in biomass rest or waste stream fractions contain these N-functionalities in proteins and free amino acids that can be used as starting materials for the synthesis of biopolymers and chemicals. This paper describes the economic and technological feasibility for cyanophycin production by fermentation of the potato waste stream Protamylasse™ or directly in plants and its subsequent conversion to a number of N-containing bulk chemicals. PMID:17876577

  8. The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

    PubMed

    Miyagi, Tatsuhiro; Tanaka, Shigeru; Hide, Izumi; Shirafuji, Toshihiko; Sakai, Norio

    2016-01-01

    G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation. PMID:26800526

  9. The transcription factor Swi4 is target for PKA regulation of cell size at the G1 to S transition in Saccharomyces cerevisiae

    PubMed Central

    Amigoni, Loredana; Colombo, Sonia; Belotti, Fiorella; Alberghina, Lilia; Martegani, Enzo

    2015-01-01

    To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G1, followed by an accumulation of cells in G2/M phase with a minimal change in the growth rate. Deletion of CLN1 gene, but not of CLN2, abolished the transient G1 phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1 gene. Transcription of CLN1 is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4 gene abolished the transient G1 arrest suggesting that Swi4 is a target responsible for PKA modulation of G1/S phase transition. We generated a SWI4 allele mutated in the consensus site for PKA (Swi4S159A) and we found that expression of Swi4S159A protein in the GG104-Swi4Δ strain did not restore the transient G1 arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1 gene expression and G1/S phase transition. PMID:26046481

  10. Acid-base titrations of functional groups on the surface of the thermophilic bacterium Anoxybacillus flavithermus: comparing a chemical equilibrium model with ATR-IR spectroscopic data.

    PubMed

    Heinrich, Hannah T M; Bremer, Phil J; Daughney, Christopher J; McQuillan, A James

    2007-02-27

    Acid-base functional groups at the surface of Anoxybacillus flavithermus (AF) were assigned from the modeling of batch titration data of bacterial suspensions and compared with those determined from in situ infrared spectroscopic titration analysis. The computer program FITMOD was used to generate a two-site Donnan model (site 1: pKa = 3.26, wet concn = 2.46 x 10(-4) mol g(-1); site 2: pKa = 6.12, wet concn = 6.55 x 10(-5) mol g(-1)), which was able to describe data for whole exponential phase cells from both batch acid-base titrations at 0.01 M ionic strength and electrophoretic mobility measurements over a range of different pH values and ionic strengths. In agreement with information on the composition of bacterial cell walls and a considerable body of modeling literature, site 1 of the model was assigned to carboxyl groups, and site 2 was assigned to amino groups. pH difference IR spectra acquired by in situ attenuated total reflection infrared (ATR-IR) spectroscopy confirmed the presence of carboxyl groups. The spectra appear to show a carboxyl pKa in the 3.3-4.0 range. Further peaks were assigned to phosphodiester groups, which deprotonated at slightly lower pH. The presence of amino groups could not be confirmed or discounted by IR spectroscopy, but a positively charged group corresponding to site 2 was implicated by electrophoretic mobility data. Carboxyl group speciation over a pH range of 2.3-10.3 at two different ionic strengths was further compared to modeling predictions. While model predictions were strongly influenced by the ionic strength change, pH difference IR data showed no significant change. This meant that modeling predictions agreed reasonably well with the IR data for 0.5 M ionic strength but not for 0.01 M ionic strength.

  11. A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway.

    PubMed

    Said, Hamid M; Ortiz, Alvaro; Ma, Thomas Y

    2003-11-01

    Vitamin B6 is essential for cellular functions and growth due to its involvement in important metabolic reactions. Humans and other mammals cannot synthesize vitamin B6 and thus must obtain this micronutrient from exogenous sources via intestinal absorption. The intestine, therefore, plays a central role in maintaining and regulating normal vitamin B6 homeostasis. Due to the water-soluble nature of vitamin B6 and the demonstration that transport of other water-soluble vitamins in intestinal epithelial cells involves specialized carrier-mediated mechanisms, we hypothesized that transport of vitamin B6 in these cells is also carrier mediated in nature. To test this hypothesis, we examined pyridoxine transport in a model system for human enterocytes, the human-derived intestinal epithelial Caco-2 cells. The results showed pyridoxine uptake to be 1) linear with time for up to 10 min of incubation and to occur with minimal metabolic alteration in the transported substrate, 2) temperature and energy dependent but Na+ independent, 3) pH dependent with higher uptake at acidic compared with alkaline pHs, 4) saturable as a function of concentration (at buffer pH 5.5 but not 7.4) with an apparent Michaelis-Menten constant (Km) of 11.99 +/- 1.41 microM and a maximal velocity (Vmax) of 67.63 +/- 3.87 pmol. mg protein-1. 3 min-1, 5) inhibited by pyridoxine structural analogs (at buffer pH 5.5 but not 7.4) but not by unrelated compounds, and 6) inhibited in a competitive manner by amiloride with an apparent inhibitor constant (Ki) of 0.39 mM. We also examined the possible regulation of pyridoxine uptake by specific intracellular regulatory pathways. The results showed that whereas modulators of PKC, Ca+2/calmodulin (CaM), and nitric oxide (NO)-mediated pathways had no effect on pyridoxine uptake, modulators of PKA-mediated pathway were found to cause significant reduction in pyridoxine uptake. This reduction was mediated via a significant inhibition in the Vmax, but not the

  12. Detection of a mitochondrial kinase complex that mediates PKA-MEK-ERK-dependent phosphorylation of mitochondrial proteins involved in the regulation of steroid biosynthesis.

    PubMed

    Paz, Cristina; Poderoso, Cecilia; Maloberti, Paula; Cornejo Maciel, Fabiana; Mendez, Carlos; Poderoso, Juan J; Podestá, Ernesto J

    2009-01-01

    In order to achieve the goal of this article, as an example we will describe the strategies followed to analyze the presence of the multi-kinase complex at the mitochondria and the posttranslational modification of two key mitochondrial proteins, which participate in the regulation of cholesterol transport across the mitochondrial membranes and in the regulation of steroid biosynthesis. Hormones, ions or growth factors modulate steroid biosynthesis by the posttranslational phosphorylation of proteins. The question still remains on how phosphorylation events transmit a specific signal to its mitochondrial site of action. Cholesterol transport requires specific interactions in mitochondria between several proteins including a multi-kinase complex. The presence of this multi-kinase complex at the mitochondria reveals the importance of the posttranslational modification of mitochondrial proteins for its activity and functions. The activation of PKA triggers the posttranslational modification of the mitochondrial acyl-CoA thioesterase (Acot2), which releases arachidonic acid (AA) in the mitochondria, and the activation of a kinase cascade that leads to the phoshorylation of the steroidogenic acute regulatory (StAR) protein. The function of StAR is to facilitate the access of cholesterol to the first enzyme of the biosynthesis process and its induction is dependent on Acot2 and intramitochondrial AA release. Truncation of the StAR protein is associated with the steroid deficiency disease, congenital lipoid adrenal hyperplasia.

  13. New Method for Determining Isotopic Values of Glutamic Acid and Phenylalanine for Estimation of Precise Trophic Position in Food Web Studies

    NASA Astrophysics Data System (ADS)

    Kamath, T.; Broek, T.; McCarthy, M.

    2012-12-01

    Compound Specific Isotope Analysis of Amino Acids (CSI-AA) has emerged as a highly precise new method of determining trophic levels of both aquatic and terrestrial organisms. Multiple studies have now shown that δ15N values for glutamic acid (Glu) and phenylalanine (Phe) can be coupled to provide extremely precise estimates of trophic position in diverse food web studies. The standard gas chromatography—isotope ratio mass spectrometer (GC-IRMS) approach is presently limited to a select number of labs since necessary equipment is both expensive and not widely accessible. Furthermore, typical GC-IRMS δ15N precision (±1‰) is significantly lower than usual bulk δ15N values (±0.1‰), thus presenting a considerable setback for precise trophic level calculations. In this study, we develop a new dual-column method to purify Glu and Phe using high performance liquid chromatography (HPLC). Phe is purified using an analytical scale reverse phase column embedded with anionic ion-pairing reagents and collected using automated fraction collection. Glu is separated from the non-polar amino acids using the same column and further purified using a hydrophilic interaction liquid chromatography (HILIC) cation and anion-exchange column and collected via automated fraction collection. Isotopic analysis of the purified AAs is then conducted on an elemental analyzer—isotope ratio mass spectrometer (EA-IRMS). As a test of this method, we present and compare the trophic position of five marine organisms—cyanobacteria, deep-sea bamboo coral, juvenile and adult white sea bass, and harbor seal, calculated using Glu and Phe δ15N values produced by both GC-IRMS and our HPLC-EA-IRMS approach. The preliminary results of this study suggest that the HPLC-EA-IRMS method is a viable alternative to GC-IRMS, which should allow accurate trophic position estimates to be made by more researchers using more readily available instrumentation.

  14. Enhancing the understanding of earthworm feeding behaviour via the use of fatty acid delta13C values determined by gas chromatography-combustion-isotope ratio mass spectrometry.

    PubMed

    Dungait, Jennifer A J; Briones, Maria J I; Bol, Roland; Evershed, Richard P

    2008-06-01

    Litter-dwelling (epigeic) Lumbricus rubellus and soil-dwelling (endogeic) Allolobophora chlorotica earthworms were observed aggregating under C(3) (delta(13)C = -31.3 per thousand; delta(15)N = 10.7 per thousand) and C(4) (delta(13)C = -12.6 per thousand; delta(15)N = 7.5 per thousand) synthetic dung pats applied to a temperate grassland (delta(13)C = -30.3 per thousand; delta(15)N = 5.7 per thousand) in an experiment carried out for 372 days. Bulk delta(13)C values of earthworms collected from beneath either C(3) or C(4) dung after 28, 56, 112 and 372 days demonstrated that (i) L. rubellus beneath C(4) dung were significantly (13)C-enriched after 56 days (delta(13)C = -23.8 per thousand) and 112 days (delta(13)C = -22.4 per thousand) compared with those from C(3) dung treatments (56 days, delta(13)C = -26.5 per thousand; 112 days, delta(13)C = -27.0 per thousand), and (ii) A. chlorotica were 2.1 per thousand (13)C-enriched (delta(13)C = -24.2 per thousand) relative to those from C(3) dung (delta(13)C = -26.3 per thousand) treatments after 372 days. Bulk delta(15)N values did not suggest significant uptake of dung N by either species beneath C(3) or C(4) dung, but showed that the endogeic species (total mean delta(15)N = 3.3 per thousand) had higher delta(15)N values than the epigeic species (total mean delta(15)N = 5.4 per thousand). Although the two species exhibited similar fatty acid profiles, individual fatty acid delta(13)C values revealed extensive routing of dietary C into body tissue of L. rubellus, but minor incorporation into A. chlorotica. In particular, the direct incorporation of microbial biomarker fatty acids (iC(17:0), aC(17:0)) from (13)C-labelled dung in situ, the routing of dung C into de novo synthesised compounds (iC(20:4)(omega)(6),C(20:5)(omega)(3), and the assimilation of essential fatty acids ((C(18:1)(omega)(9), C(18:1)(omega(7), C(18:2)(omega(6), C(18:3)(omega)(3)) derived from dung, were determined.

  15. Comparison of the concentration-effect relationship of a local antiinflammatory agent and oral acetylsalicylic acid: the value of local application.

    PubMed

    Poisson, M; Ralambosoa, C; Blehaut, H; Astoin, J

    1985-01-01

    Using a pharmacological model, the comparison between acetylsalicylic acid (ASA), administered orally, and a solution combining two salicylate derivatives (ethyl 5-methoxy-salicylate and 3-phenyl-propyl-salicylate), applied locally, demonstrated the value of the local application. Indeed, the pharmacological activity was highly significant and directly related to the tissue concentration of salicyl ions, which was higher after local application of the solution than after oral administration of ASA. The local solution also resulted in a lower plasma concentration of salicylate ions, allowing high plasma salicylate concentrations to be avoided. PMID:4074414

  16. Selective expression of a dominant-negative type Iα PKA regulatory subunit in striatal medium spiny neurons impairs gene expression and leads to reduced feeding and locomotor activity.

    PubMed

    Yang, Linghai; Gilbert, Merle L; Zheng, Ruimao; McKnight, G Stanley

    2014-04-01

    Striatal medium spiny neurons (MSNs) mediate many of the physiological effects of dopamine, including the regulation of feeding and motor behaviors. Dopaminergic inputs from the midbrain modulate MSN excitability through pathways that involve cAMP and protein kinase A (PKA), but the physiological role of specific PKA isoforms in MSN neurons remains poorly understood. One of the major PKA regulatory (R) subunit isoforms expressed in MSNs is RIIβ, which localizes the PKA holoenzyme primarily to dendrites by interaction with AKAP5 and other scaffolding proteins. However, RI (RIα and RIβ) subunits are also expressed in MSNs and the RI holoenzyme has a weaker affinity for most scaffolding proteins and tends to localize in the cell body. We generated mice with selective expression of a dominant-negative RI subunit (RIαB) in striatal MSNs and show that this dominant-negative RIαB localizes to the cytoplasm and specifically inhibits type I PKA activity in the striatum. These mice are normal at birth; however, soon after weaning they exhibit growth retardation and the adult mice are hypophagic, lean, and resistant to high-fat diet-induced hyperphagia and obesity. The RIαB-expressing mice also exhibit decreased locomotor activity and decreased dopamine-regulated CREB phosphorylation and c-fos gene expression in the striatum. Our results demonstrate a critical role for cytoplasmic RI-PKA holoenzyme in gene regulation and the overall physiological function of MSNs. PMID:24695708

  17. A Kinase Anchoring Protein 9 Is a Novel Myosin VI Binding Partner That Links Myosin VI with the PKA Pathway in Myogenic Cells.

    PubMed

    Karolczak, Justyna; Sobczak, Magdalena; Skowronek, Krzysztof; Rędowicz, Maria Jolanta

    2015-01-01

    Myosin VI (MVI) is a unique motor protein moving towards the minus end of actin filaments unlike other known myosins. Its important role has recently been postulated for striated muscle and myogenic cells. Since MVI functions through interactions of C-terminal globular tail (GT) domain with tissue specific partners, we performed a search for MVI partners in myoblasts and myotubes using affinity chromatography with GST-tagged MVI-GT domain as a bait. A kinase anchoring protein 9 (AKAP9), a regulator of PKA activity, was identified by means of mass spectrometry as a possible MVI interacting partner both in undifferentiated and differentiating myoblasts and in myotubes. Coimmunoprecipitation and proximity ligation assay confirmed that both proteins could interact. MVI and AKAP9 colocalized at Rab5 containing early endosomes. Similarly to MVI, the amount of AKAP9 decreased during myoblast differentiation. However, in MVI-depleted cells, both cAMP and PKA levels were increased and a change in the MVI motor-dependent AKAP9 distribution was observed. Moreover, we found that PKA phosphorylated MVI-GT domain, thus implying functional relevance of MVI-AKAP9 interaction. We postulate that this novel interaction linking MVI with the PKA pathway could be important for targeting AKAP9-PKA complex within cells and/or providing PKA to phosphorylate MVI tail domain.

  18. Acid-Base Formalism Extended to Excited State for O-H···S Hydrogen Bonding Interaction.

    PubMed

    Bhattacharyya, Surjendu; Roy, Ved Prakash; Wategaonkar, Sanjay

    2016-09-01

    Hydrogen bond can be regarded as an interaction between a base and a proton covalently bound to another base. In this context the strength of hydrogen bond scales with the proton affinity of the acceptor base and the pKa of the donor, i.e., it follows the acid-base formalism. This has been amply demonstrated in conventional hydrogen bonds. Is this also true for the unconventional hydrogen bonds involving lesser electronegative elements such as sulfur atom? In our previous work, we had established that the strength of O-H···S hydrogen bonding (HB) interaction scales with the proton affinity (PA) of the acceptor. In this work, we have investigated the other counterpart, i.e., the H-bonding interaction between the photoacids with different pKa values with a common base such as the H2O and H2S. The 1:1 complexes of five para substituted phenols p-aminophenol, p-cresol, p-fluorophenol, p-chlorophenol, and p-cyanophenol with H2O and H2S were investigated experimentally and computationally. The investigations were also extended to the excited states. The experimental observations of the spectral shifts in the O-H stretching frequency and the S1-S0 band origins were correlated with the pKa of the donors. Ab initio calculations at the MP2 and various dispersion corrected density functional levels of theory were performed to compute the dissociation energy (D0) of the complexes. The quantum theory of atoms in molecules (QTAIM), noncovalent interaction (NCI) method, natural bonding orbital (NBO) analysis, and natural decomposition analysis (NEDA) were carried out for further characterization of HB interaction. The O-H stretching frequency red shifts and the dissociation energies were found to be lower for the O-H···S hydrogen bonded systems compared to those for the O-H···O H-bound systems. Despite being dominated by the dispersion interaction the O-H···S interaction in the H2S complexes also conformed to the acid-base formalism, i.e., the D0 and the O-H red shift

  19. Acid-Base Formalism Extended to Excited State for O-H···S Hydrogen Bonding Interaction.

    PubMed

    Bhattacharyya, Surjendu; Roy, Ved Prakash; Wategaonkar, Sanjay

    2016-09-01

    Hydrogen bond can be regarded as an interaction between a base and a proton covalently bound to another base. In this context the strength of hydrogen bond scales with the proton affinity of the acceptor base and the pKa of the donor, i.e., it follows the acid-base formalism. This has been amply demonstrated in conventional hydrogen bonds. Is this also true for the unconventional hydrogen bonds involving lesser electronegative elements such as sulfur atom? In our previous work, we had established that the strength of O-H···S hydrogen bonding (HB) interaction scales with the proton affinity (PA) of the acceptor. In this work, we have investigated the other counterpart, i.e., the H-bonding interaction between the photoacids with different pKa values with a common base such as the H2O and H2S. The 1:1 complexes of five para substituted phenols p-aminophenol, p-cresol, p-fluorophenol, p-chlorophenol, and p-cyanophenol with H2O and H2S were investigated experimentally and computationally. The investigations were also extended to the excited states. The experimental observations of the spectral shifts in the O-H stretching frequency and the S1-S0 band origins were correlated with the pKa of the donors. Ab initio calculations at the MP2 and various dispersion corrected density functional levels of theory were performed to compute the dissociation energy (D0) of the complexes. The quantum theory of atoms in molecules (QTAIM), noncovalent interaction (NCI) method, natural bonding orbital (NBO) analysis, and natural decomposition analysis (NEDA) were carried out for further characterization of HB interaction. The O-H stretching frequency red shifts and the dissociation energies were found to be lower for the O-H···S hydrogen bonded systems compared to those for the O-H···O H-bound systems. Despite being dominated by the dispersion interaction the O-H···S interaction in the H2S complexes also conformed to the acid-base formalism, i.e., the D0 and the O-H red shift

  20. cAMP-PKA phosphorylation of tau confers risk for degeneration in aging association cortex

    PubMed Central

    Carlyle, Becky C.; Nairn, Angus C.; Wang, Min; Yang, Yang; Jin, Lu E.; Simen, Arthur A.; Ramos, Brian P.; Bordner, Kelly A.; Craft, George E.; Davies, Peter; Pletikos, Mihovil; Šestan, Nenad; Arnsten, Amy F. T.; Paspalas, Constantinos D.

    2014-01-01

    The pattern of neurodegeneration in Alzheimer’s disease (AD) is very distinctive: neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau selectively affect pyramidal neurons of the aging association cortex that interconnect extensively through glutamate synapses on dendritic spines. In contrast, primary sensory cortices have few NFTs, even in late-stage disease. Understanding this selective vulnerability, and why advancing age is such a high risk factor for the degenerative process, may help to reveal disease etiology and provide targets for intervention. Our study has revealed age-related increase in cAMP-dependent protein kinase (PKA) phosphorylation of tau at serine 214 (pS214-tau) in monkey dorsolateral prefrontal association cortex (dlPFC), which specifically targets spine synapses and the Ca2+-storing spine apparatus. This increase is mirrored by loss of phosphodiesterase 4A from the spine apparatus, consistent with increase in cAMP-Ca2+ signaling in aging spines. Phosphorylated tau was not detected in primary visual cortex, similar to the pattern observed in AD. We also report electron microscopic evidence of previously unidentified vesicular trafficking of phosphorylated tau in normal association cortex—in axons in young dlPFC vs. in spines in aged dlPFC—consistent with the transneuronal lesion spread reported in genetic rodent models. pS214-Tau was not observed in normal aged mice, suggesting that it arises with the evolutionary expansion of corticocortical connections in primates, crossing the threshold into NFTs and degeneration in humans. Thus, the cAMP-Ca2+ signaling mechanisms, needed for flexibly modulating network strength in young association cortex, confer vulnerability to degeneration when dysregulated with advancing age. PMID:24707050

  1. Improving the nutritional value of crops through enhancement of L-ascorbic acid (vitamin C) content: rationale and biotechnological opportunities.

    PubMed

    Hancock, Robert D; Viola, Roberto

    2005-06-29

    L-Ascorbic acid (AsA, vitamin C) is an essential human nutrient that must be obtained in the diet, with the vast majority being obtained from plant foods. A vitamin C-deficient diet results in the onset of scurvy, which can have lethal consequences. However, vitamin C has also been implicated in the prevention of chronic diseases such as heart disease, stroke, cancer, and several neurodegenerative diseases. Although the supporting evidence for these claims is disputed, the dietary allowances for vitamin C have been recently increased in several countries, including the United States. This scenario, together with the general perception by consumers of vitamin C as being of benefit in the prevention of several lifestyle diseases and associated with general "well-being", contributes to a market rationale for enhancing the vitamin C content of crops. In recent years, there has been substantial progress in the understanding of vitamin C biochemistry in plants with a number of structural genes cloned. Here these findings are reviewed, and a description of how such knowledge could be applied to the nutritional enhancement of crops is given. PMID:15969504

  2. The Value of PIC Cystography in Detecting De Novo and Residual Vesicoureteral Reflux after Dextranomer/Hyaluronic Acid Copolymer Injection.

    PubMed

    Palmer, B W; Hemphill, M; Wettengel, K; Kropp, B P; Frimberger, D

    2011-01-01

    The endoscopic injection of Dx/HA in the management of vesicoureteral reflux (VUR) has become an accepted alternative to open surgery. In the current study we evaluated the value of cystography to detect de novo contralateral VUR in unilateral cases of VUR at the time of Dx/HA injection and correlated the findings of immediate post-Dx/HA injection cystography during the same anesthesia to 2-month postoperative VCUG to evaluate its ability to predict successful surgical outcomes. The current study aimed to evaluate whether an intraoperatively performed cystogram could replace postoperative studies. But a negative intraoperative cystogram correlates with the postoperative study in only 80%. Considering the 75-80% success rate of Dx/HA implantation, the addition of intraoperative cystograms cannot replace postoperative studies. In patients treated with unilateral VUR, PIC cystography can detect occult VUR and prevent postoperative contralateral new onset of VUR.

  3. Chemical rescue, multiple ionizable groups, and general acid-base catalysis in the HDV genomic ribozyme.

    PubMed

    Perrotta, Anne T; Wadkins, Timothy S; Been, Michael D

    2006-07-01

    In the ribozyme from the hepatitis delta virus (HDV) genomic strand RNA, a cytosine side chain is proposed to facilitate proton transfer in the transition state of the reaction and, thus, act as a general acid-base catalyst. Mutation of this active-site cytosine (C75) reduced RNA cleavage rates by as much as one million-fold, but addition of exogenous cytosine and certain nucleobase or imidazole analogs can partially rescue activity in these mutants. However, pH-rate profiles for the rescued reactions were bell shaped, and only one leg of the pH-rate curve could be attributed to ionization of the exogenous nucleobase or buffer. When a second potential ionizable nucleobase (C41) was removed, one leg of the bell-shaped curve was eliminated in the chemical-rescue reaction. With this construct, the apparent pK(a) determined from the pH-rate profile correlated with the solution pK(a) of the buffer, and the contribution of the buffer to the rate enhancement could be directly evaluated in a free-energy or Brønsted plot. The free-energy relationship between the acid dissociation constant of the buffer and the rate constant for cleavage (Brønsted value, beta, = approximately 0.5) was consistent with a mechanism in which the buffer acted as a general acid-base catalyst. These data support the hypothesis that cytosine 75, in the intact ribozyme, acts as a general acid-base catalyst.

  4. Investigation of mono/competitive adsorption of environmentally relevant ionized weak acids on graphite: impact of molecular properties and thermodynamics.

    PubMed

    Moustafa, Ahmed M A; McPhedran, Kerry N; Moreira, Jesús; Gamal El-Din, Mohamed

    2014-12-16

    The thermodynamics of adsorption and competitive interactions of five weak acids on a graphite surface was assessed in alkaline solutions. Adsorption of the acids in mono- and multicompound solutions followed their Freundlich isotherms which suggest a diversity of graphite adsorption sites as confirmed by the presence of carboxylic and phenolic groups observed on graphite surfaces. Thermodynamic calculations assigned the formation of the negatively charged assisted hydrogen bond (-CAHB) between ionized solutes and adsorbent surface groups as the possible adsorption mechanism. However, the similar pKa values of current acids resulted in comparable free energies for -CAHB formation (ΔG(-CAHB)) being less than solvation free energies (ΔGSolv). Thus, additional ΔG is supplemented by increased hydrophobicity due to proton exchange of ionized acids with water (ΔΔG Hydrophobicity). Adsorption capacities and competition coefficients indicated that ΔΔG Hydrophobicity values depend on the neutral and ionized acid Kow. Competitive adsorption implies that multilayer adsorption may occur via hydrophobic bonding with the CH3 ends of the self-assembled layer which affects the acid adsorption capacities in mixtures as compared to monocompound solutions. The determination of adsorption mechanisms will assist in understanding of the fate and bioavailability of emerging and classical weak acids released into natural waters.

  5. Determination of dissociation constants of aristolochic acid I and II by capillary electrophoresis with carboxymethyl chitosan-coated capillary.

    PubMed

    Fu, Xiaofang; Liu, Yi; Li, Wei; Bai, Yu; Liao, Yiping; Liu, Huwei

    2011-07-15

    Aristolochic acid-I and aristolochic acid-II have been proved to be the main bioactive and toxic component in Aristolochia plants. As a result, the determination of their dissociation constants, which are important property parameters for weak acids, is highly desired for related pharmacological and toxicological studies. In this work, the dissociation constant values of aristolochic acid-I and aristolochic acid-II were determined by capillary electrophoresis using carboxymethyl chitosan-coated capillary, based on their electrophoretic mobilities by using nonlinear regression as well as linear regression, showing that the two models give comparable results. The data were also compared with those obtained by capillary electrophoresis with polybrene-coated capillary, and no conspicuous difference was observed. The correlation coefficients were all higher than 0.998 for both linear and nonlinear regression model. The pKa values were found to be 3.3±0.1 for aristolochic acid-I and 3.2±0.1 for aristolochic acid-II.

  6. The effects of structural variations of thiophene-containing Ru(II) complexes on the acid-base and DNA binding properties.

    PubMed

    Yuan, Cui-Li; Zhang, An-Guo; Zheng, Ze-Bo; Wang, Ke-Zhi

    2013-03-01

    A phenylthiophenyl-bearing Ru(II) complex of [Ru(bpy)₂(Hbptip)](PF₆)₂ {bpy = 2,2'-bipyridine, Hbptip = 2-(4-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesized and characterized by elemental analysis, ¹H NMR spectroscopy, and electrospray ionization mass spectrometry. The ground- and excited-state acid-base properties of the complex were studied by UV-visible absorption and photoluminescence spectrophotometric pH titrations and the negative logarithm values of the ground-state acid ionization constants were derived to be pK(a1) = 1.31 ± 0.09 and pK(a2) = 5.71 ± 0.11 with the pK(a2) associated deprotonation/protonation process occurring over 3 pK(a) units more acidic than thiophenyl-free parent complex of [Ru(bpy)₂(Hpip)]²⁺ {Hpip = 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline}. The calf thymus DNA-binding properties of [Ru(bpy)₂(Hbptip)]²⁺ in Tris-HCl buffer (pH 7.1 and 50 mM NaCl) were investigated by DNA viscosities and density functional theoretical calculations as well as UV-visible and emission spectroscopy techniques of UV-visible and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]⁴⁻, DNA competitive binding with ethidium bromide, DNA melting experiments, and reverse salt effects. The complex was evidenced to bind to the DNA intercalatively with binding affinity being greater than those for previously reported analogs of [Ru(bpy)₂(Hip)]²⁺, [Ru(bpy)₂(Htip)]²⁺, and [Ru(bpy)₂(Haptip)]²⁺ {Hip = 1H-imidazo[4,5-f][1,10]phenanthroline, Htip = 2-thiophenimidazo[4,5-f][1,10]phenanthroline, Haptip = 2-(5-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}.

  7. 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

  8. Transmission of electronic effects through the {closo-1-CB9} and {closo-1-CB11} cages: apparent dissociation constants for series of [closo-1-CB9H8-1-COOH-10-X] and [closo-1-CB11H10-1-COOH-12-X] acids.

    PubMed

    Pecyna, Jacek G; Ringstrand, Bryan; Kaszyński, Piotr

    2012-05-01

    The apparent ionization constants pK(a)' for series of carboxylic acids [closo-1-CB(9)H(8)-1-COOH-10-X](-) (1) and [closo-1-CB(11)H(10)-1-COOH-12-X](-) (2), where X = H, I, n-C(6)H(13), (+)NMe(3), (+)N(2), (+)SMe(2), OC(5)H(11), were measured in EtOH/H(2)O (1/1, v/v) at 24 °C. Correlation analysis of the pK(a)' values using Hammett substituent constants σ(p)(X) gave the reaction constant ρ = 0.87 ± 0.04 for series 1 and ρ = 1.00 ± 0.09 for series 2. These values are higher than for derivatives of PhCH═CHCOOH (ρ = 0.70 ± 0.09 in 55% EtOH) and correspond to 56% and 65% efficiencies in transmission of electronic effects by [closo-1-CB(9)H(10)](-) (E) and [closo-1-CB(11)H(12)](-) (F), respectively, as compared to benzene (A). Experimental results were supported with DFT calculations of relative acidity for series of acids derived from A, E, and F in aqueous medium.

  9. Determination of thermodynamic values of acidic dissociation constants and complexation constants of profens and their utilization for optimization of separation conditions by Simul 5 Complex.

    PubMed

    Riesová, Martina; Svobodová, Jana; Ušelová, Kateřina; Tošner, Zdeněk; Zusková, Iva; Gaš, Bohuslav

    2014-10-17

    In this paper we determine acid dissociation constants, limiting ionic mobilities, complexation constants with β-cyclodextrin or heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, and mobilities of resulting complexes of profens, using capillary zone electrophoresis and affinity capillary electrophoresis. Complexation parameters are determined for both neutral and fully charged forms of profens and further corrected for actual ionic strength and variable viscosity in order to obtain thermodynamic values of complexation constants. The accuracy of obtained complexation parameters is verified by multidimensional nonlinear regression of affinity capillary electrophoretic data, which provides the acid dissociation and complexation parameters within one set of measurements, and by NMR technique. A good agreement among all discussed methods was obtained. Determined complexation parameters were used as input parameters for simulations of electrophoretic separation of profens by Simul 5 Complex. An excellent agreement of experimental and simulated results was achieved in terms of positions, shapes, and amplitudes of analyte peaks, confirming the applicability of Simul 5 Complex to complex systems, and accuracy of obtained physical-chemical constants. Simultaneously, we were able to demonstrate the influence of electromigration dispersion on the separation efficiency, which is not possible using the common theoretical approaches, and predict the electromigration order reversals of profen peaks. We have shown that determined acid dissociation and complexation parameters in combination with tool Simul 5 Complex software can be used for optimization of separation conditions in capillary electrophoresis.

  10. A unique quantitative method of acid value of edible oils and studying the impact of heating on edible oils by UV-Vis spectrometry.

    PubMed

    Zhang, Wenle; Li, Na; Feng, Yuyan; Su, Shujun; Li, Tao; Liang, Bing

    2015-10-15

    UV-Vis spectroscopy coupled with chemometrics was used effectively to study the impact of heating on edible oils (corn oil, sunflower oil, rapeseed oil, peanut oil, soybean oil and sesame oil) and determine their acid value. Analysis of their first derivative spectra showed that the peak at 370 nm was a common indicator of the heated oils. Partial least squares regression (PLS) and principle component regression (PCR) were applied to building individual quantitative models of acid value for each kind of oil, respectively. The PLS models had a better performance than PCR models, with determination coefficients (R(2)) of 0.9904-0.9977 and root mean square errors (RMSE) of 0.0230-0.0794 for the prediction sets of each kind of oil, respectively. An integrate quantitative model built by support vector regression for all the six kinds of oils was also developed and gave a satisfactory prediction with a R(2) of 0.9932 and a RMSE of 0.0656.

  11. Potential-Dependent Electrocatalytic Pathways: Controlling Reactivity with pKa for Mechanistic Investigation of a Nickel-Based Hydrogen Evolution Catalyst.

    PubMed

    Rountree, Eric S; Dempsey, Jillian L

    2015-10-21

    A detailed mechanistic analysis is presented for the hydrogen evolution catalyst [Ni(P2(Ph)N2(Ph))2(CH2CN)][BF4]2 in acetonitrile (P2(Ph)N2(Ph) = 1,3,5,7-tetraphenyl-1,5-diaza-3,7-diphosphacyclooctane). This complex has a Ni(II/I) redox couple at −0.83 V and a Ni(I/0) redox couple at −1.03 V versus Fc(+/0). These two closely spaced redox events both promote proton reduction catalysis, each via a distinct mechanism: an electrochemical ECEC pathway and an EECC route. The EECC mechanism, operative at more negative potentials, was isolated through use of a weak acid (anilinium, pKa = 10.6 in CH3CN) to avert protonation of the singly reduced species. Electroanalytical methods and time-resolved spectroscopy were used to analyze the kinetics of the elementary steps of hydrogen evolution catalysis. The rate constant for the formation of a nickel(II)–hydride intermediate was determined via measurements of peak shift (k1 = 1.2 × 106 M(-1) s(-1)) and through foot-of-the-wave analysis (k1 = 6.5 × 106 M(-1) s(-1)). Reactivity of the isolated hydride with acid to release hydrogen and regenerate the nickel(II) complex was monitored by stopped-flow spectroscopy. Kinetics obtained from stopped-flow measurements are corroborated by current plateau analysis of the catalytic cyclic voltammograms. These kinetic data suggest the presence of an off-cycle intermediate in the reaction.

  12. PKA, Rap1, ERK1/2, and p90RSK mediate PGE2 and EP4 signaling in neonatal ventricular myocytes.

    PubMed

    He, Quan; Harding, Pamela; LaPointe, Margot C

    2010-01-01

    We have previously reported that 1) inhibition of cyclooxygenase-2 and PGE(2) production reduces hypertrophy after myocardial infarction in mice and 2) PGE(2) acting through its EP4 receptor causes hypertrophy of neonatal ventricular myocytes (NVMs) via ERK1/2. It is known that EP4 couples to adenylate cyclase, cAMP, and PKA. The present study was designed to determine interactions between the cAMP-PKA pathway and ERK1/2 and to further characterize events downstream of ERK1/2. We hypothesized that PKA and the small GTPase Rap are upstream of ERK1/2 and that 90-kDa ribosomal S6 kinase (p90RSK) is activated downstream. Treatment of NVMs with PGE(2) activated Rap, and this activation was inhibited in part by an EP4 antagonist and PKA inhibition. Transfection of a dominant negative mutant of Rap reduced PGE(2) activation of ERK1/2. PGE(2) activation of p90RSK was also dependent on EP4, PKA, and Rap. We also tested the involvement of Rap, ERK1/2, and p90RSK in PGE(2) regulation of gene expression. PGE(2) stimulation of brain natriuretic peptide promoter activity was blocked by either ERK1/2 inhibition or a dominant negative mutation of p90RSK. PGE(2) stimulation of c-Fos was dependent on EP4, PKA, ERK1/2, and p90RSK, whereas only the latter two kinases were involved in PGE(2) regulation of early growth response-1. Finally, we tested the involvement of EP4-dependent signaling in the NVM growth response and found that the overexpression of EP4 increased NVM cell size. We conclude that EP4-dependent signaling in NVMs in part involves PKA, Rap, ERK1/2, and p90RSK and results in the increased expression of brain natriuretic peptide and c-Fos.

  13. Calcitonin gene-related peptide inhibits autophagic-lysosomal proteolysis through cAMP/PKA signaling in rat skeletal muscles.

    PubMed

    Machado, Juliano; Manfredi, Leandro H; Silveira, Wilian A; Gonçalves, Dawit A P; Lustrino, Danilo; Zanon, Neusa M; Kettelhut, Isis C; Navegantes, Luiz C

    2016-03-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide released by motor neuron in skeletal muscle and modulates the neuromuscular transmission by induction of synthesis and insertion of acetylcholine receptor on postsynaptic muscle membrane; however, its role in skeletal muscle protein metabolism remains unclear. We examined the in vitro and in vivo effects of CGRP on protein breakdown and signaling pathways in control skeletal muscles and muscles following denervation (DEN) in rats. In isolated muscles, CGRP (10(-10) to 10(-6)M) reduced basal and DEN-induced activation of overall proteolysis in a concentration-dependent manner. The in vitro anti-proteolytic effect of CGRP was completely abolished by CGRP8-37, a CGRP receptor antagonist. CGRP down-regulated the lysosomal proteolysis, the mRNA levels of LC3b, Gabarapl1 and cathepsin L and the protein content of LC3-II in control and denervated muscles. In parallel, CGRP elevated cAMP levels, stimulated PKA/CREB signaling and increased Foxo1 phosphorylation in both conditions. In denervated muscles and starved C2C12 cells, Rp-8-Br-cAMPs or PKI, two PKA inhibitors, completely abolished the inhibitory effect of CGRP on Foxo1, 3 and 4 and LC3 lipidation. A single injection of CGRP (100 μg kg(-1)) in denervated rats increased the phosphorylation levels of CREB and Akt, inhibited Foxo transcriptional activity, the LC3 lipidation as well as the mRNA levels of LC3b and cathepsin L, two bona fide targets of Foxo. This study shows for the first time that CGRP exerts a direct inhibitory action on autophagic-lysosomal proteolysis in control and denervated skeletal muscle by recruiting cAMP/PKA signaling, effects that are related to inhibition of Foxo activity and LC3 lipidation. PMID:26718975

  14. Quantitative aspects of electrolysis in electromembrane extractions of acidic and basic analytes.

    PubMed

    Šlampová, Andrea; Kubáň, Pavel; Boček, Petr

    2015-08-01

    Electrolysis is omnipresent in all electrochemical processes including electromembrane extraction (EME). The effects of electrolysis on quantitative aspects of EME were comprehensively evaluated for a set of acidic (substituted phenols) and basic (basic drugs) analytes. EMEs were carried out across supported liquid membranes formed by 1-ethyl-2-nitrobenzene at standard EME conditions, i.e., acidic analytes were extracted from alkaline into alkaline solutions and basic analytes were extracted from acidic into acidic solutions. Electric potential applied across the EME systems was 50 V and extraction recoveries of analytes as well as pH values of donor and acceptor solutions were determined after each EME. It has been proven that electrolysis plays a more significant role than has ever been thought before in EME. Electrolytically produced H(+) and OH(-) ions had a significant effect on pH values of acceptor solutions and variations of up to 8.5 pH units were obtained at standard EME conditions. pH values of donor solutions were affected only negligibly due to their significantly higher volumes. The observed variations in pH values of acceptor solutions had fatal consequences on quantitative EME results of weak and medium strong acidic/basic analytes. A direct relation was observed between the decrease in extraction recoveries of the analytes, their pKa values and the acceptor solution pH values. Acceptor solutions consisting of high concentrations of weak bases or acids were thus proposed as suitable EME operational solutions since they efficiently eliminated the electrolytically induced pH variations, offered stable EME performances and were easily compatible with subsequent analytical methods.

  15. Quantitative aspects of electrolysis in electromembrane extractions of acidic and basic analytes.

    PubMed

    Šlampová, Andrea; Kubáň, Pavel; Boček, Petr

    2015-08-01

    Electrolysis is omnipresent in all electrochemical processes including electromembrane extraction (EME). The effects of electrolysis on quantitative aspects of EME were comprehensively evaluated for a set of acidic (substituted phenols) and basic (basic drugs) analytes. EMEs were carried out across supported liquid membranes formed by 1-ethyl-2-nitrobenzene at standard EME conditions, i.e., acidic analytes were extracted from alkaline into alkaline solutions and basic analytes were extracted from acidic into acidic solutions. Electric potential applied across the EME systems was 50 V and extraction recoveries of analytes as well as pH values of donor and acceptor solutions were determined after each EME. It has been proven that electrolysis plays a more significant role than has ever been thought before in EME. Electrolytically produced H(+) and OH(-) ions had a significant effect on pH values of acceptor solutions and variations of up to 8.5 pH units were obtained at standard EME conditions. pH values of donor solutions were affected only negligibly due to their significantly higher volumes. The observed variations in pH values of acceptor solutions had fatal consequences on quantitative EME results of weak and medium strong acidic/basic analytes. A direct relation was observed between the decrease in extraction recoveries of the analytes, their pKa values and the acceptor solution pH values. Acceptor solutions consisting of high concentrations of weak bases or acids were thus proposed as suitable EME operational solutions since they efficiently eliminated the electrolytically induced pH variations, offered stable EME performances and were easily compatible with subsequent analytical methods. PMID:26320790

  16. Mechanism of the AppABLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 pKa on the Forward and Reverse Ground-State Reactions.

    PubMed

    Gil, Agnieszka A; Haigney, Allison; Laptenok, Sergey P; Brust, Richard; Lukacs, Andras; Iuliano, James N; Jeng, Jessica; Melief, Eduard H; Zhao, Rui-Kun; Yoon, EunBin; Clark, Ian P; Towrie, Michael; Greetham, Gregory M; Ng, Annabelle; Truglio, James J; French, Jarrod B; Meech, Stephen R; Tonge, Peter J

    2016-01-27

    The transcriptional antirepressor AppA is a blue light using flavin (BLUF) photoreceptor that releases the transcriptional repressor PpsR upon photoexcitation. Light activation of AppA involves changes in a hydrogen-bonding network that surrounds the flavin chromophore on the nanosecond time scale, while the dark state of AppA is then recovered in a light-independent reaction with a dramatically longer half-life of 15 min. Residue Y21, a component of the hydrogen-bonding network, is known to be essential for photoactivity. Here, we directly explore the effect of the Y21 pKa on dark state recovery by replacing Y21 with fluorotyrosine analogues that increase the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements confirm that the structure of AppA is unperturbed by fluorotyrosine substitution, and that there is a small (3-fold) change in the photokinetics of the forward reaction over the fluorotyrosine series. However, reduction of 3.5 pH units in the pKa of Y21 increases the rate of dark state recovery by 4000-fold with a Brønsted coefficient of ∼ 1, indicating that the Y21 proton is completely transferred in the transition state leading from light to dark adapted AppA. A large solvent isotope effect of ∼ 6-8 is also observed on the rate of dark state recovery. These data establish that the acidity of Y21 is a crucial factor for stabilizing the light activated form of the protein, and have been used to propose a model for dark state recovery that will ultimately prove useful for tuning the properties of BLUF photosensors for optogenetic applications. PMID:26708408

  17. The biochemistry of citric acid accumulation by Aspergillus niger.

    PubMed

    Karaffa, L; Sándor, E; Fekete, E; Szentirmai, A

    2001-01-01

    Fungi, in particular Aspergilli, are well known for their potential to overproduce a variety of organic acids. These microorganisms have an intrinsic ability to accumulate these substances and it is generally believed that this provides the fungi with an ecological advantage, since they grow rather well at pH 3 to 5, while some species even tolerate pH values as low as 1.5. Organic acid production can be stimulated and in a number of cases conditions have been found that result in almost quantitative conversion of carbon substrate into acid. This is exploited in large-scale production of a number of organic acids like citric-, gluconic- and itaconic acid. Both in production volume as well as in knowledge available, citrate is by far the major organic acid. Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid) is a true bulk product with an estimated global production of over 900 thousand tons in the year 2000. Till the beginning of the 20th century, it was exclusively extracted from lemons. Since the global market was dominated by an Italian cartel, other means of production were sought. Chemical synthesis was possible, but not suitable due to expensive raw materials and a complicated process with low yield. The discovery of citrate accumulation by Aspergillus niger led to a rapid development of a fermentation process, which only a decade later accounted for a large part of the global production. The application of citric acid is based on three of its properties: (1) acidity and buffer capacity, (2) taste and flavour, and (3) chelation of metal ions. Because of its three acid groups with pKa values of 3.1, 4.7 and 6.4, citrate is able to produce a very low pH in solution, but is also useful as a buffer over a broad range of pH values (2 to 7). Citric acid has a pleasant acid taste which leaves little aftertaste. It sometimes enhances flavour, but is also able to mask sweetness, such as the aspartame taste in diet beverages. Chelation of metal ions is a very

  18. Simulations of acid dissociation constants of polyprotic acids in near-critical and supercritical water.

    PubMed

    Halstead, Simon J; An, Pengna

    2013-12-01

    This paper reports a molecular dynamics study on the dissociation of sulfuric acid and phosphoric acid in near-critical and supercritical water. pK(a) is known to vary as the temperature and pressure vary, and this variation has important implications for corrosion in supercritical water reaction vessels. This work uses the SPC/E water model and solutes based upon DFT calculations to examine both structural and thermodynamic properties of the dissociation processes. An increase in solute-solvent ordering is observed for larger charges, and this also corresponds to a lower rate of diffusion. All dissociation reactions become less favored with increasing temperature except pK(a1) for sulfuric acid which becomes significantly more favored until 748 K.

  19. Which factors determine the acidity of the phytochromobilin chromophore of plant phytochrome?

    PubMed

    Anders Borg, O; Durbeej, Bo

    2008-05-14

    Quantum chemical calculations aimed at identifying the factors controlling the acidity of phytochromobilin, the tetrapyrrole chromophore of the plant photoreceptor phytochrome, are reported. Phytochrome is converted from an inactive (Pr) to an active form (Pfr) through a series of events initiated by a Z --> E photoisomerization of phytochromobilin, forming the Lumi-R intermediate, and much controversy exists as to whether the protonation state of the chromophore (cationic in Pr with all nitrogens protonated) changes during the photoactivation. Here, relative ground (S0) and excited-state (S1) pKa s of all four pyrrole moieties of phytochromobilin in all 64 possible configurations with respect to the three methine bridges are calculated in a protein-like environment, using a recently benchmarked level of theory. Accordingly, the relationships between acidity and chromophore geometry and charge distribution, hydrogen bonding, and light absorption are investigated in some detail, and discussed in terms of possible mechanisms making a proton transfer reaction more probable along the Pr --> Pfr reaction than in the parent cationic Pr state. It is found that charge distribution in the cationic species, intra-molecular hydrogen bonding in the neutral, and hydrogen bonding with two highly conserved aspartate and histidine residues have a significant effect on the acidity, while overall chromophore geometry and electronic state are less important factors. Furthermore, based on the calculations, two processes that may facilitate a proton transfer by substantially lowering the pKa s relative to their Pr values are identified: (i) a thermal Z,anti --> Z,syn isomerization at C5, occurring after formation of Lumi-R; (ii) a perturbation of the hydrogen bonding network which in Pr comprises the nitrogens of pyrroles A, B and C and the two aspartate and histidine residues.

  20. Cytoplasmic anchoring of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs) is required for meiotic arrest of porcine full-grown and growing oocytes.

    PubMed

    Nishimura, Takanori; Fujii, Wataru; Sugiura, Koji; Naito, Kunihiko

    2014-03-01

    Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence. PMID:24501172

  1. The Ras/PKA signaling pathway may control RNA polymerase II elongation via the Spt4p/Spt5p complex in Saccharomyces cerevisiae.

    PubMed Central

    Howard, Susie C; Hester, Arelis; Herman, Paul K

    2003-01-01

    The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription. PMID:14668364

  2. Suppression of LPS-Induced TNF-α Production in Macrophages by cAMP Is Mediated by PKA-AKAP95-p105

    PubMed Central

    Wall, Estelle A.; Zavzavadjian, Joelle R.; Chang, Mi Sook; Randhawa, Baljinder; Zhu, Xiaocui; Hsueh, Robert C.; Liu, Jamie; Driver, Adrienne; Bao, Xiaoyan Robert; Sternweis, Paul C.; Simon, Melvin I.; Fraser, Iain D. C.

    2009-01-01

    The activation of macrophages through Toll-like receptor (TLR) pathways leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The inflammatory potential of this response can be reduced by compounds, such as prostaglandin E2, that induce the production of cyclic adenosine monophosphate (cAMP). Through experiments with cAMP analogs and multigene RNA interference (RNAi), we showed that key anti-inflammatory effects of cAMP were mediated specifically by cAMP-dependent protein kinase (PKA). Selective inhibitors of PKA anchoring, time-lapse microscopy, and RNAi screening suggested that differential mechanisms of PKA action existed. We showed a specific role for A kinase-anchoring protein 95 in suppressing the expression of the gene encoding tumor necrosis factor-α, which involved phosphorylation of p105 (also known as Nfkb1) by PKA at a site adjacent to the region targeted by inhibitor of nuclear factor κB kinases. These data suggest that crosstalk between the TLR4 and cAMP pathways in macrophages can be coordinated through PKA-dependent scaffolds that localize specific pools of the kinase to distinct substrates. PMID:19531803

  3. Phosphorylation of Ser1166 on GluN2B by PKA Is Critical to Synaptic NMDA Receptor Function and Ca2+ Signaling in Spines

    PubMed Central

    Murphy, Jessica A.; Stein, Ivar S.; Lau, C. Geoffrey; Peixoto, Rui T.; Aman, Teresa K.; Kaneko, Naoki; Aromolaran, Kelly; Saulnier, Jessica L.; Popescu, Gabriela K.

    2014-01-01

    The NMDA-type glutamate receptor (NMDAR) is essential for synaptogenesis, synaptic plasticity, and higher cognitive function. Emerging evidence indicates that NMDAR Ca2+ permeability is under the control of cAMP/protein kinase A (PKA) signaling. Whereas the functional impact of PKA on NMDAR-dependent Ca2+ signaling is well established, the molecular target remains unknown. Here we identify serine residue 1166 (Ser1166) in the carboxy-terminal tail of the NMDAR subunit GluN2B to be a direct molecular and functional target of PKA phosphorylation critical to NMDAR-dependent Ca2+ permeation and Ca2+ signaling in spines. Activation of β-adrenergic and D1/D5-dopamine receptors induces Ser1166 phosphorylation. Loss of this single phosphorylation site abolishes PKA-dependent potentiation of NMDAR Ca2+ permeation, synaptic currents, and Ca2+ rises in dendritic spines. We further show that adverse experience in the form of forced swim, but not exposure to fox urine, elicits striking phosphorylation of Ser1166 in vivo, indicating differential impact of different forms of stress. Our data identify a novel molecular and functional target of PKA essential to NMDAR-mediated Ca2+ signaling at synapses and regulated by the emotional response to stress. PMID:24431445

  4. A PKA-Csk-pp60Src signaling pathway regulates the switch between endothelial cell invasion and cell-cell adhesion during vascular sprouting

    PubMed Central

    Jin, Hui; Garmy-Susini, Barbara; Avraamides, Christie J.; Stoletov, Konstantin; Klemke, Richard L.

    2010-01-01

    Angiogenesis is controlled by signals that stimulate motility in endothelial cells at the tips of vascular sprouts while maintaining cell-cell adhesion in the stalks of angiogenic sprouts. We show here that Gs-linked G protein–coupled receptor activation of cAMP-dependent protein kinase (PKA) plays an important role in regulating the switch between endothelial cell adhesion and migration by activating C-terminal Src kinase, leading to inhibition of pp60Src. Activated PKA blocks pp60Src-dependent vascular endot helial-cadherin phosphorylation, thereby stimulating cell-cell adhesion while suppressing endothelial cell polarization, motility, angiogenesis, and vascular permeability. Similar to the actions of Notch and Dll4, PKA activation blocks sprouting in newly forming embryonic blood vessels, while PKA inhibition promotes excessive sprouting in these vessels. These findings demonstrate that G protein–coupled receptors and PKA regulate vascular sprouting during angiogenesis by controlling endothelial cell migration and cell-cell adhesion through their actions on pp60Src. PMID:20826718

  5. A PKA-Csk-pp60Src signaling pathway regulates the switch between endothelial cell invasion and cell-cell adhesion during vascular sprouting.

    PubMed

    Jin, Hui; Garmy-Susini, Barbara; Avraamides, Christie J; Stoletov, Konstantin; Klemke, Richard L; Varner, Judith A

    2010-12-16

    Angiogenesis is controlled by signals that stimulate motility in endothelial cells at the tips of vascular sprouts while maintaining cell-cell adhesion in the stalks of angiogenic sprouts. We show here that Gs-linked G protein-coupled receptor activation of cAMP-dependent protein kinase (PKA) plays an important role in regulating the switch between endothelial cell adhesion and migration by activating C-terminal Src kinase, leading to inhibition of pp60Src. Activated PKA blocks pp60Src-dependent vascular endot helial-cadherin phosphorylation, thereby stimulating cell-cell adhesion while suppressing endothelial cell polarization, motility, angiogenesis, and vascular permeability. Similar to the actions of Notch and Dll4, PKA activation blocks sprouting in newly forming embryonic blood vessels, while PKA inhibition promotes excessive sprouting in these vessels. These findings demonstrate that G protein-coupled receptors and PKA regulate vascular sprouting during angiogenesis by controlling endothelial cell migration and cell-cell adhesion through their actions on pp60Src. PMID:20826718

  6. Galacturonic Acid Inhibits the Growth of Saccharomyces cerevisiae on Galactose, Xylose, and Arabinose

    PubMed Central

    Huisjes, Eline H.; de Hulster, Erik; van Dam, Jan C.; Pronk, Jack T.

    2012-01-01

    The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pKa value of galacturonic acid (3.51), the addition of 10 g · liter−1 galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter−1 galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks. PMID:22582063

  7. Galacturonic acid inhibits the growth of Saccharomyces cerevisiae on galactose, xylose, and arabinose.

    PubMed

    Huisjes, Eline H; de Hulster, Erik; van Dam, Jan C; Pronk, Jack T; van Maris, Antonius J A

    2012-08-01

    The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pK(a) value of galacturonic acid (3.51), the addition of 10 g · liter(-1) galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter(-1) galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks. PMID:22582063

  8. Galacturonic acid inhibits the growth of Saccharomyces cerevisiae on galactose, xylose, and arabinose.

    PubMed

    Huisjes, Eline H; de Hulster, Erik; van Dam, Jan C; Pronk, Jack T; van Maris, Antonius J A

    2012-08-01

    The efficient fermentation of mixed substrates is essential for the microbial conversion of second-generation feedstocks, including pectin-rich waste streams such as citrus peel and sugar beet pulp. Galacturonic acid is a major constituent of hydrolysates of these pectin-rich materials. The yeast Saccharomyces cerevisiae, the main producer of bioethanol, cannot use this sugar acid. The impact of galacturonic acid on alcoholic fermentation by S. cerevisiae was investigated with anaerobic batch cultures grown on mixtures of glucose and galactose at various galacturonic acid concentrations and on a mixture of glucose, xylose, and arabinose. In cultures grown at pH 5.0, which is well above the pK(a) value of galacturonic acid (3.51), the addition of 10 g · liter(-1) galacturonic acid did not affect galactose fermentation kinetics and growth. In cultures grown at pH 3.5, the addition of 10 g · liter(-1) galacturonic acid did not significantly affect glucose consumption. However, at this lower pH, galacturonic acid completely inhibited growth on galactose and reduced galactose consumption rates by 87%. Additionally, it was shown that galacturonic acid strongly inhibits the fermentation of xylose and arabinose by the engineered pentose-fermenting S. cerevisiae strain IMS0010. The data indicate that inhibition occurs when nondissociated galacturonic acid is present extracellularly and corroborate the hypothesis that a combination of a decreased substrate uptake rate due to competitive inhibition on Gal2p, an increased energy requirement to maintain cellular homeostasis, and/or an accumulation of galacturonic acid 1-phosphate contributes to the inhibition. The role of galacturonic acid as an inhibitor of sugar fermentation should be considered in the design of yeast fermentation processes based on pectin-rich feedstocks.

  9. General Base-General Acid Catalysis in Human Histone Deacetylase 8.

    PubMed

    Gantt, Sister M Lucy; Decroos, Christophe; Lee, Matthew S; Gullett, Laura E; Bowman, Christine M; Christianson, David W; Fierke, Carol A

    2016-02-01

    Histone deacetylases (HDACs) regulate cellular processes such as differentiation and apoptosis and are targeted by anticancer therapeutics in development and in the clinic. HDAC8 is a metal-dependent class I HDAC and is proposed to use a general acid-base catalytic pair in the mechanism of amide bond hydrolysis. Here, we report site-directed mutagenesis and enzymological measurements to elucidate the catalytic mechanism of HDAC8. Specifically, we focus on the catalytic function of Y306 and the histidine-aspartate dyads H142-D176 and H143-D183. Additionally, we report X-ray crystal structures of four representative HDAC8 mutants: D176N, D176N/Y306F, D176A/Y306F, and H142A/Y306F. These structures provide a useful framework for understanding enzymological measurements. The pH dependence of kcat/KM for wild-type Co(II)-HDAC8 is bell-shaped with two pKa values of 7.4 and 10.0. The upper pKa reflects the ionization of the metal-bound water molecule and shifts to 9.1 in Zn(II)-HDAC8. The H142A mutant has activity 230-fold lower than that of wild-type HDAC8, but the pKa1 value is not altered. Y306F HDAC8 is 150-fold less active than the wild-type enzyme; crystal structures show that Y306 hydrogen bonds with the zinc-bound substrate carbonyl, poised for transition state stabilization. The H143A and H142A/H143A mutants exhibit activity that is >80000-fold lower than that of wild-type HDAC8; the buried D176N and D176A mutants have significant catalytic effects, with more subtle effects caused by D183N and D183A. These enzymological and structural studies strongly suggest that H143 functions as a single general base-general acid catalyst, while H142 remains positively charged and serves as an electrostatic catalyst for transition state stabilization.

  10. An acidity scale of tetrafluoroborate