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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. PMID:25089727

  2. 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

  3. Arginine: Its pKa value revisited

    PubMed Central

    Fitch, Carolyn A; Platzer, Gerald; Okon, Mark; Garcia-Moreno E, Bertrand; McIntosh, Lawrence P

    2015-01-01

    Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pKa value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pKa value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pKa value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group. PMID:25808204

  4. Arginine: Its pKa value revisited.

    PubMed

    Fitch, Carolyn A; Platzer, Gerald; Okon, Mark; Garcia-Moreno, Bertrand E; McIntosh, Lawrence P

    2015-05-01

    Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pKa value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼ 12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pKa value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pKa value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group. PMID:25808204

  5. 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

  6. The pKa values of acidic and basic residues buried at the same internal location in a protein are governed by different factors

    PubMed Central

    Harms, Michael J.; Castañeda, Carlos A.; Schlessman, Jamie L.; Sue, Gloria R.; Bertrand García-Moreno, E

    2009-01-01

    Summary The pKa values of internal ionizable groups are usually very different than the normal pKa values of ionizable groups in water. To examine the molecular determinants of pKa values of internal groups, we compared the properties of Lys, Asp and Glu at internal position 38 in staphylococcal nuclease. Lys-38 titrates with a normal or elevated pKa whereas Asp-38 and Glu-38 titrate with elevated pKa values of 7.0 and 7.2, respectively. In the structure of the L38K variant, the buried amino group of the Lys-38 side chain makes an ion pair with Glu-122; whereas, in the structure of the L38E variant, the buried carboxyl group of Glu-38 interacts with two backbone amides and has several nearby carboxyl oxygen atoms. Previously we showed that the pKa of Lys-38 is normal owing to structural reorganization and water penetration concomitant with ionization of the Lys side chain. In contrast, the pKa of Asp-38 and Glu-38 are perturbed significantly owing to an imbalance between favorable polar interactions and unfavorable contributions from dehydration and from Coulomb interactions with surface carboxylic groups. Their ionization is also coupled to subtle structural reorganization. These results illustrate the complex interplay between local polarity, Coulomb interactions and structural reorganization as determinants of pKa values of internal groups in proteins. This study suggests that improvements to computational methods for pKa calculations will require explicit treatment of the conformational reorganization that can occur when internal groups ionize. PMID:19324049

  7. Rapid Calculation of Protein pKa Values Using Rosetta

    PubMed Central

    Kilambi, Krishna Praneeth; Gray, Jeffrey J.

    2012-01-01

    We developed a Rosetta-based Monte Carlo method to calculate the pKa values of protein residues that commonly exhibit variable protonation states (Asp, Glu, Lys, His, and Tyr). We tested the technique by calculating pKa values for 264 residues from 34 proteins. The standard Rosetta score function, which is independent of any environmental conditions, failed to capture pKa shifts. After incorporating a Coulomb electrostatic potential and optimizing the solvation reference energies for pKa calculations, we employed a method that allowed side-chain flexibility and achieved a root mean-square deviation (RMSD) of 0.83 from experimental values (0.68 after discounting 11 predictions with an error over 2 pH units). Additional degrees of side-chain conformational freedom for the proximal residues facilitated the capture of charge-charge interactions in a few cases, resulting in an overall RMSD of 0.85 pH units. The addition of backbone flexibility increased the overall RMSD to 0.93 pH units but improved relative pKa predictions for proximal catalytic residues. The method also captures large pKa shifts of lysine and some glutamate point mutations in staphylococcal nuclease. Thus, a simple and fast method based on the Rosetta score function and limited conformational sampling produces pKa values that will be useful when rapid estimation is essential, such as in docking, design, and folding. PMID:22947875

  8. 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.

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

    PubMed

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

    2014-03-01

    This article investigates an ensemble-based technique called Bayesian Model Averaging (BMA) to improve the performance of protein amino acid pKa predictions. Structure-based pKa 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 pKa prediction, ranging from empirical statistical models to ab initio quantum mechanical approaches. However, each of these methods are based on a set of conceptual assumptions that can effect a model's accuracy and generalizability for pKa 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 García-Moreno lab. Our cross-validation study demonstrates that the aggregated estimate obtained from BMA outperforms all individual prediction methods with improvements ranging from 45 to 73% over other method classes. This study also compares BMA's predictive performance to other ensemble-based techniques and demonstrates that BMA can outperform these approaches with improvements ranging from 27 to 60%. This work illustrates a new possible mechanism for improving the accuracy of pKa prediction and lays the foundation for future work on aggregate models that balance computational cost with prediction accuracy. PMID:23946048

  10. NMR Determination of Protein pKa Values in the Solid State

    PubMed Central

    Schmidt, Heather L. Frericks; Shah, Gautam J.; Sperling, Lindsay J.; Rienstra, Chad M.

    2010-01-01

    Charged residues play an important role in defining key mechanistic features in many biomolecules. Determining the pKa values of large, membrane or fibrillar proteins can be challenging with traditional methods. In this study we show how solid-state NMR is used to monitor chemical shift changes during a pH titration for the small soluble β1 immunoglobulin binding domain of protein G. The chemical shifts of all the amino acids with charged side-chains throughout the uniformly-13C,15N-labeled protein were monitored over several samples varying in pH; pKa values were determined from these shifts for E27, D36, and E42, and the bounds for the pKa of other acidic side-chain resonances were determined. Additionally, this study shows how the calculated pKa values give insights into the crystal packing of the protein. PMID:20563223

  11. Bayesian Model Aggregation for Ensemble-Based Estimates of Protein pKa Values

    PubMed Central

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

    2013-01-01

    This paper investigates an ensemble-based technique called Bayesian Model Averaging (BMA) to improve the performance of protein amino acid pKa predictions. Structure-based pKa 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 pKa prediction, ranging from empirical statistical models to ab initio quantum mechanical approaches. However, each of these methods are based on a set of conceptual assumptions that can effect a model’s accuracy and generalizability for pKa 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 García-Moreno lab. Our cross-validation study demonstrates that the aggregated estimate obtained from BMA outperforms all individual prediction methods with improvements ranging from 45-73% over other method classes. This study also compares BMA’s predictive performance to other ensemble-based techniques and demonstrates that BMA can outperform these approaches with improvements ranging from 27-60%. This work illustrates a new possible mechanism for improving the accuracy of pKa prediction and lays the foundation for future work on aggregate models that balance computational cost with prediction accuracy. PMID:23946048

  12. NMR determination of pKa values in α-synuclein

    PubMed Central

    Croke, Robyn L; Patil, Sharadrao M; Quevreaux, Jason; Kendall, Debra A; Alexandrescu, Andrei T

    2011-01-01

    The intrinsically unfolded protein α-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pKa values for all 26 sites in the protein that ionize below pH 7 using 2D 1H-15N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pKa values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pKa values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For α-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pKa of His50, a residue of particular interest since it is flanked within one turn of the α-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pKa of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pKa shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pKa values of uncomplexed α-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding. PMID:21280118

  13. 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)

  14. The pKa Cooperative: A Collaborative Effort to Advance Structure-Based Calculations of pKa values and Electrostatic Effects in Proteins

    PubMed Central

    Nielsen, Jens E.; Gunner, M. R.; Bertrand García-Moreno, E.

    2012-01-01

    The pKa Cooperative http://www.pkacoop.org was organized to advance development of accurate and useful computational methods for structure-based calculation of pKa values and electrostatic energy in proteins. The Cooperative brings together laboratories with expertise and interest in theoretical, computational and experimental studies of protein electrostatics. To improve structure-based energy calculations it is necessary to better understand the physical character and molecular determinants of electrostatic effects. The Cooperative thus intends to foment experimental research into fundamental aspects of proteins that depend on electrostatic interactions. It will maintain a depository for experimental data useful for critical assessment of methods for structure-based electrostatics calculations. To help guide the development of computational methods the Cooperative will organize blind prediction exercises. As a first step, computational laboratories were invited to reproduce an unpublished set of experimental pKa values of acidic and basic residues introduced in the interior of staphylococcal nuclease by site-directed mutagenesis. The pKa values of these groups are unique and challenging to simulate owing to the large magnitude of their shifts relative to normal pKa values in water. Many computational methods were tested in this 1st Blind Prediction Challenge and critical assessment exercise. A workshop was organized in the Telluride Science Research Center to assess objectively the performance of many computational methods tested on this one extensive dataset. This volume of PROTEINS: Structure, Function, and Bioinformatics introduces the pKa Cooperative, presents reports submitted by participants in the blind prediction challenge, and highlights some of the problems in structure-based calculations identified during this exercise. PMID:22002877

  15. The pKa Cooperative: a collaborative effort to advance structure-based calculations of pKa values and electrostatic effects in proteins.

    PubMed

    Nielsen, Jens E; Gunner, M R; García-Moreno, Bertrand E

    2011-12-01

    The pK(a) Cooperative (http://www.pkacoop.org) was organized to advance development of accurate and useful computational methods for structure-based calculation of pK(a) values and electrostatic energies in proteins. The Cooperative brings together laboratories with expertise and interest in theoretical, computational, and experimental studies of protein electrostatics. To improve structure-based energy calculations, it is necessary to better understand the physical character and molecular determinants of electrostatic effects. Thus, the Cooperative intends to foment experimental research into fundamental aspects of proteins that depend on electrostatic interactions. It will maintain a depository for experimental data useful for critical assessment of methods for structure-based electrostatics calculations. To help guide the development of computational methods, the Cooperative will organize blind prediction exercises. As a first step, computational laboratories were invited to reproduce an unpublished set of experimental pK(a) values of acidic and basic residues introduced in the interior of staphylococcal nuclease by site-directed mutagenesis. The pK(a) values of these groups are unique and challenging to simulate owing to the large magnitude of their shifts relative to normal pK(a) values in water. Many computational methods were tested in this first Blind Prediction Challenge and critical assessment exercise. A workshop was organized in the Telluride Science Research Center to objectively assess the performance of many computational methods tested on this one extensive data set. This volume of Proteins: Structure, Function, and Bioinformatics introduces the pK(a) Cooperative, presents reports submitted by participants in the Blind Prediction Challenge, and highlights some of the problems in structure-based calculations identified during this exercise. PMID:22002877

  16. Determinants of the pKa values of ionizable residues in an intrinsically disordered protein.

    PubMed

    Neira, José L; Rizzuti, Bruno; Iovanna, Juan L

    2016-05-15

    Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes; in humans, they are often associated with diseases. The protein NUPR1 is a multifunctional IDP involved in the development and progression of pancreatic cancer; therefore, it constitutes a target for drug design. In an effort to contribute to the understanding of the conformational features of NUPR1 and to provide clues on amino acid interactions in disordered states of proteins, we measured the pKa values of all its acidic groups (aspartic and glutamic residues, and backbone C terminus) by using NMR spectroscopy at low (100 mM) and high (500 mM) NaCl concentration. At low ionic strength, the pKa values were similar to those reported for random-coil models, except for Glu18 and Asp19, suggesting electrostatic interactions around these residues. Molecular modelling and simulation indicate an additional, significant role of nearby proline residues in determining the polypeptide conformational features and water accessibility in the region around Glu18, modulating the titration properties of these amino acids. In the other acidic residues of NUPR1, the small deviations of pKa values (compared to those expected for a random-coil) are likely due to electrostatic interactions with charged adjacent residues, which should be reduced at high NaCl concentrations. In fact, at high ionic strength, the pKa values of the aspartic residues were similar to those in a random coil, but there were still small differences for those of glutamic acids, probably due to hydrogen-bond formation. The overall findings suggest that local interactions and hydrophobic effects play a major role in determining the electrostatic features of NUPR1, whereas long-range charge contributions appear to be of lesser importance. PMID:27046343

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

    PubMed

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

    2016-04-20

    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

  18. 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

  19. 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

  20. pKa prediction for acidic phosphorus-containing compounds using multiple linear regression with computational descriptors.

    PubMed

    Yu, Donghai; Du, Ruobing; Xiao, Ji-Chang

    2016-07-01

    Ninety-six acidic phosphorus-containing molecules with pKa 1.88 to 6.26 were collected and divided into training and test sets by random sampling. Structural parameters were obtained by density functional theory calculation of the molecules. The relationship between the experimental pKa values and structural parameters was obtained by multiple linear regression fitting for the training set, and tested with the test set; the R(2) values were 0.974 and 0.966 for the training and test sets, respectively. This regression equation, which quantitatively describes the influence of structural parameters on pKa , and can be used to predict pKa values of similar structures, is significant for the design of new acidic phosphorus-containing extractants. © 2016 Wiley Periodicals, Inc. PMID:27218266

  1. Theoretical prediction of relative and absolute pKa values of aminopyridines.

    PubMed

    Caballero, N A; Melendez, F J; Muñoz-Caro, C; Niño, A

    2006-11-20

    This work presents a study aimed at the theoretical prediction of pK(a) values of aminopyridines, as a factor responsible for the activity of these compounds as blockers of the voltage-dependent K(+) channels. To cover a large range of pK(a) values, a total of seven substituted pyridines is considered as a calibration set: pyridine, 2-aminopyridine, 3-aminopyridine, 4-aminopyridine, 2-chloropyridine, 3-chloropyridine, and 4-methylpirydine. Using ab initio G1, G2 and G3 extrapolation methods, and the CPCM variant of the Polarizable Continuum Model for solvation, we calculate gas phase and solvation free energies. pK(a) values are obtained from these data using a thermodynamic cycle for describing protonation in aqueous and gas phases. The results show that the relatively inexpensive G1 level of theory is the most accurate at predicting pK(a) values in aminopyridines. The highest standard deviation with respect to the experimental data is 0.69 pK(a) units for absolute values calculations. The difference increases slightly to 0.74 pK(a) units when the pK(a) is computed relative to the pyridine molecule. Considering only compounds at least as basic as pyridine (the values of interest for bioactive aminopyridines) the error falls to 0.10 and 0.12 pK(a) units for the absolute and relative computations, respectively. The technique can be used to predict the effect of electronegative substituents in the pK(a) of 4-AP, the most active aminopyridine considered in this work. Thus, 2-chloro and 3-chloro-4-aminopyridine are taken into account. The results show a decrease of the pK(a), suggesting that these compounds are less active than 4-AP at blocking the K(+) channel. PMID:16844281

  2. 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

  3. Review: Bilirubin pKa studies; new models and theories indicate high pKa values in water, dimethylformamide and DMSO

    PubMed Central

    2010-01-01

    Background Correct aqueous pKa values of unconjugated bilirubin (UCB), a poorly-soluble, unstable substance, are essential for understanding its functions. Our prior solvent partition studies, of unlabeled and [14C] UCB, indicated pKa values above 8.0. These high values were attributed to effects of internal H-bonding in UCB. Many earlier and subsequent studies have reported lower pKa values, some even below 5.0, which are often used to describe the behavior of UCB. We here review 18 published studies that assessed aqueous pKa values of UCB, critically evaluating their methodologies in relation to essential preconditions for valid pKa measurements (short-duration experiments with purified UCB below saturation and accounting for self-association of UCB). Results These re-assessments identified major deficiencies that invalidate the results of all but our partition studies. New theoretical modeling of UCB titrations shows remarkable, unexpected effects of self-association, yielding falsely low pKa estimates, and provides some rationalization of the titration anomalies. The titration behavior reported for a soluble thioether conjugate of UCB at high aqueous concentrations is shown to be highly anomalous. Theoretical re-interpretations of data in DMSO and dimethylformamide show that those indirectly-derived aqueous pKa values are unacceptable, and indicate new, high average pKa values for UCB in non-aqueous media (>11 in DMSO and, probably, >10 in dimethylformamide). Conclusions No reliable aqueous pKa values of UCB are available for comparison with our partition-derived results. A companion paper shows that only the high pKa values can explain the pH-dependence of UCB binding to phospholipids, cyclodextrins, and alkyl-glycoside and bile salt micelles. PMID:20350304

  4. Progress in the prediction of pKa values in proteins.

    PubMed

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

    2011-12-01

    The pK(a) -cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pK(a) values and protein electrostatics in general. The first round of the pK(a) -cooperative, which challenged computational labs to carry out blind predictions against pK(a) s experimentally determined in the laboratory of Bertrand Garcia-Moreno, was completed and results discussed at the Telluride meeting (July 6-10, 2009). This article 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 pK(a) calculations, emphasizing methods that were used by the participants in calculating the blind pK(a) 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 pK(a) calculations. PMID:22002859

  5. 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

  6. Characterisation of selected active agents regarding pKa values, solubility concentrations and pH profiles by SiriusT3.

    PubMed

    Schönherr, D; Wollatz, U; Haznar-Garbacz, D; Hanke, U; Box, K J; Taylor, R; Ruiz, R; Beato, S; Becker, D; Weitschies, W

    2015-05-01

    The aim of this work was to determine pKa values and solubility properties of 34active agents using the SiriusT3 apparatus. The selected drug substances belong to the groups of ACE-inhibitors, β-blockers, antidiabetics and lipid lowering substances. Experimentally obtained pKa and intrinsic solubility values were compared to calculated values (program ACD/ChemSketch) and pKa values to published data as well. Solubility-pH profiles were generated to visualise the substance solubility over the gastrointestinal pH range. The relationship between the solubility characteristic of a substance, its bioavailability and categorisation according to the Biopharmaceutics Classification System (BCS) was examined as well. The results showed a good agreement between experimentally obtained, calculated and published pKa values. The measured and calculated intrinsic solubility values indicated several major deviations. All solubility-pH profiles showed the expected shape and appearance for acids, bases or zwitterionic substances. The obtained results for the pKa and solubility measurements of the examined active agents may help to predict their physicochemical behaviour in vivo, and to understand the bioavailability of the substances according to their BCS categorisation. The easy and reproducible determination of pKa and solubility values makes the SiriusT3 apparatus a useful tool in early stages of drug and formulation development. PMID:25758123

  7. Development of Methods for the Determination of pKa Values.

    PubMed

    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

  8. 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

  9. 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

  10. 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.

  11. 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. PMID:24952470

  12. pKa values in proteins determined by electrostatics applied to molecular dynamics trajectories.

    PubMed

    Meyer, Tim; Knapp, Ernst-Walter

    2015-06-01

    For a benchmark set of 194 measured pKa values in 13 proteins, electrostatic energy computations are performed in which pKa values are computed by solving the Poisson-Boltzmann equation. In contrast to the previous approach of Karlsberg(+) (KB(+)) that essentially used protein crystal structures with variations in their side chain conformations, the present approach (KB2(+)MD) uses protein conformations from four molecular dynamics (MD) simulations of 10 ns each. These MD simulations are performed with different specific but fixed protonation patterns, selected to sample the conformational space for the different protonation patterns faithfully. The root-mean-square deviation between computed and measured pKa values (pKa RMSD) is shown to be reduced from 1.17 pH units using KB(+) to 0.96 pH units using KB2(+)MD. The pKa RMSD can be further reduced to 0.79 pH units, if each conformation is energy-minimized with a dielectric constant of εmin = 4 prior to calculating the electrostatic energy. The electrostatic energy expressions upon which the computations are based have been reformulated such that they do not involve terms that mix protein and solvent environment contributions and no thermodynamic cycle is needed. As a consequence, conformations of the titratable residues can be treated independently in the protein and solvent environments. In addition, the energy terms used here avoid the so-called intrinsic pKa and can therefore be interpreted without reference to arbitrary protonation states and conformations. PMID:26575575

  13. pKa prediction using group philicity.

    PubMed

    Parthasarathi, R; Padmanabhan, J; Elango, M; Chitra, K; Subramanian, V; Chattaraj, P K

    2006-05-25

    Acid-base dissociation constants (pK(a) values) are important in understanding the chemical, environmental and toxicological properties of molecules. Though various methods have been developed to predict pK(a) by experimental and theoretical models, prediction of pK(a) is still complicated. Hence, a new approach for predicting pK(a) using the group philicity concept has been attempted. Presence of known functional groups in a molecule is utilized as the most important indicator to predict pK(a). The power of this descriptor in describing pK(a) for the series of carboxylic acids, various substituted phenols, anilines, phosphoric acids, and alcohols is probed. Results reveal that the group electrophilicity is suitable for effectively predicting the pK(a) values. PMID:16706412

  14. Large shifts in pKa values of lysine residues buried inside a protein

    PubMed Central

    Isom, Daniel G.; Castañeda, Carlos A.; Cannon, Brian R.; García-Moreno E., Bertrand

    2011-01-01

    Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pKa values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pKa values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H+ transport. The pKa values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pKa values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function. PMID:21389271

  15. Large shifts in pKa values of lysine residues buried inside a protein.

    PubMed

    Isom, Daniel G; Castañeda, Carlos A; Cannon, Brian R; García-Moreno, Bertrand

    2011-03-29

    Internal ionizable groups in proteins are relatively rare but they are essential for catalysis and energy transduction. To examine molecular determinants of their unusual and functionally important properties, we engineered 25 variants of staphylococcal nuclease with lysine residues at internal positions. Nineteen of the Lys residues have depressed pK(a) values, some as low as 5.3, and 20 titrate without triggering any detectable conformational reorganization. Apparently, simply by being buried in the protein interior, these Lys residues acquired pK(a) values comparable to those of naturally occurring internal ionizable groups involved in catalysis and biological H(+) transport. The pK(a) values of some of the internal Lys residues were affected by interactions with surface carboxylic groups. The apparent polarizability reported by the pK(a) values varied significantly from location to location inside the protein. These data will enable an unprecedented examination of the positional dependence of the dielectric response of a protein. This study also shows that the ability of proteins to withstand the presence of charges in their hydrophobic interior is a fundamental property inherent to all stable proteins, not a specialized adaptation unique to proteins that evolved to depend on internal charges for function. PMID:21389271

  16. PROGRESS IN THE PREDICTION OF pKa VALUES IN PROTEINS

    PubMed Central

    Baker, Nathan; Baptista, Antonio; Huang, Yong; Milletti, Francesca; Nielsen, Jens Erik; Farrell, Damien; Carstensen, Tommy; Olsson, Mats H. M.; Shen, Jana K.; Warwicker, Jim; Williams, Sarah; Word, J. Michael

    2011-01-01

    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. PMID:22002859

  17. 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

  18. Is the prediction of pKa values by constant-pH molecular dynamics being hindered by inherited problems?

    PubMed

    Machuqueiro, Miguel; Baptista, António M

    2011-12-01

    In this study, we investigate two factors that can hinder the performance of constant-pH molecular dynamics methods in predicting protein pK(a) values, using hen egg white lysozyme as a test system. The first factor is related to the molecular definition and pK(a) value of model compounds in the Poisson-Boltzmann framework. We address this by defining the model compound as a molecular fragment with an associated pK(a) value that is calibrated against experimental data, which results in a decrease of 0.12 units in pK(a) errors. The second addressed factor is the possibility that detrimental structural distortions are being introduced in the simulations by the underlying molecular mechanics force field. This issue is investigated by analyzing how the gradual structural rearrangements affect the predicted pK(a) values. The two GROMOS force fields studied here (43A1 and 53A6) yield good pK(a) predictions, although a time-dependent performance is observed: 43A1 performs better after a few nanoseconds of structural reorganization (pK(a) errors of ~0.45), while 53A6 gives the best prediction right at the first nanosecond (pK(a) errors of 0.42). These results suggest that the good performance of constant-pH molecular dynamics methods could be further improved if these force field limitations were overcome. PMID:22072522

  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. PMID:26592414

  20. Solubilization of amphiphilic carboxylic acids in nonionic micelles: determination of partition coefficients from pKa measurements and NMR experiments.

    PubMed

    Dupont-Leclercq, Laurence; Giroux, Sébastien; Henry, Bernard; Rubini, Patrice

    2007-10-01

    The solubilization of octylamidotartaric acid (C8T) and octanoic acid (C8C) in Triton X-100 and Brij 58 nonionic micelles has been studied by pHmetric and 1H NMR self-diffusion experiments. As both C8C and C8T exhibit acid-base properties, a distinction between the partition of the neutral acidic form, in terms of the partition coefficient KPH, and the partition of the charged basic form, in terms of the partition coefficient KP-, has been made. The acidity constants, Ka, of C8T and C8C in the presence of micelles have been evaluated from pHmetric experiments. For both solutes, an increase in the pKa is observed in micellar media due to the difference in the partition of acidic and basic forms of the solutes. A model has been developed to determine KPH and KP- from the pKa shifts observed. The values obtained by this pKa shift modeling method and those from self-diffusion coefficient measurements are in good agreement. The acidic form of C8C is incorporated to a larger extent into the Brij 58 micelles than the acidic form of C8T, whereas the opposite trend is observed for the basic forms. Both the acidic and basic forms of C8T are more easily incorporated into Brij 58 micelles than into Triton X-100 micelles. The influence of the structure of the polar head on the solubilization properties is demonstrated. Moreover, evidence for the localization of the solutes in the micelles is obtained from the comparison of the partition coefficients and from 1H NMR data. PMID:17850105

  1. The pKa of Brønsted acids controls their reactivity with diazo compounds.

    PubMed

    Fei, Na; Sauter, Basilius; Gillingham, Dennis

    2016-06-14

    We study the O-alkylation of phosphate groups by alkyl diazo compounds in a range of small molecules and biopolymers. We show that the relatively high pKa of phosphate in comparison to the other naturally occurring Brønsted acids can be exploited to control alkylation selectivity. We provide a simple protocol for chemical modification of some of the most important instances of phosphates in natural compounds including in small molecule metabolites, nucleic acids, and peptides. PMID:27212133

  2. 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

  3. 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

  4. Predicting the stability of aprotic solvents in Li-air batteries: pKa calculations of aliphatic C-H acids in dimethyl sulfoxide

    NASA Astrophysics Data System (ADS)

    Bryantsev, Vyacheslav S.

    2013-02-01

    Superoxide is a strong base that can induce base-catalyzed autoxidation of weakly acidic solvents. We report on the performance of several computational protocols for predicting pKa values for a wide range of aliphatic C-H acids in DMSO. Calculations at the MP2/CBS level with CCSD(T)/aug-cc-pVDZ corrections and solvent effects calculated using the SVPE model provide the best overall performance (rms deviation is 0.65 pKa). The B3LYP, M06, and M06-2X functionals can also achieve high accuracy (<1 pKa) by employing empirical corrections to fit the experimental data. Computational results provide a convenient means of screening for suitable solvents in Li-air batteries.

  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 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.

  7. Determination of pKa values of 2-amino-2-oxazolines by capillary electrophoresis.

    PubMed

    Matoga, M; Laborde-Kummer, E; Langlois, M H; Dallet, P; Bosc, J J; Jarry, C; Dubost, J P

    2003-01-17

    The dissociation constants of new 2-amino-2-oxazolines were determined by capillary electrophoresis (CE) as a new technique. A method based on a linear model has been used in the CE determination. A series of eight 2-amino-2-oxazolines are investigated to determine their ionization constant. Among them, three new oxazolines synthesized are presented. The Ka values were obtained from the plots of reciprocal effective mobility against inverse concentrations of protons. The potentiometric method (PM) was performed as a comparative method. No significant differences were observed between the determined dissociation constants using both methods. Thus, the pKa values have been found to vary between 8.55 and 8.68. PMID:12564697

  8. Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration.

    PubMed Central

    Fitch, Carolyn A; Karp, Daniel A; Lee, Kelly K; Stites, Wesley E; Lattman, Eaton E; García-Moreno E, Bertrand

    2002-01-01

    Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups. PMID:12023252

  9. pH titration studies of an SH2 domain-phosphopeptide complex: unusual histidine and phosphate pKa values.

    PubMed Central

    Singer, A. U.; Forman-Kay, J. D.

    1997-01-01

    Electrostatic interactions in a complex of the phospholipase C-gamma 1 C-terminal SH2 domain with a high-affinity binding phosphopeptide representing the sequence around Tyr 1021 of the beta platelet-derived growth factor receptor were studied by pKa determination of various titratable groups over the pH range of 5 to 8. A histidine residue that is highly conserved among SH2 domains (His beta D4) and the phosphotyrosine (pTyr) phosphate group show pKa values significantly lower than average for these residue types in proteins. The reduced pKa of these two groups is due to the proximity of the highly positively charged pTyr binding pocket. The unusual pKa of His beta D4 is also due to burial from solvent in a hydrogen-bonding network that appears necessary for the positioning of arginine residues involved in pTyr binding. Mutation of the analogous histidine in other SH2 domains has been shown to abrogate pTyr binding. In addition to these large shifts in pKa values, smaller effects were observed for the titratable groups of a glutamic acid and histidine near the C-terminus of the the second helix due to its helical dipole. Finally, exchange behavior of arginine guanidinium protons with solvent as a function of pH in this SH2 domain-phosphopeptide complex confirms previous descriptions of the roles of different arginines in the structure and function of this protein. PMID:9300491

  10. 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.

  11. pKa Values for the Unfolded State under Native Conditions Explain the pH-Dependent Stability of PGB1

    PubMed Central

    Lindman, Stina; Bauer, Mikael C.; Lund, Mikael; Diehl, Carl; Mulder, Frans A.A.; Akke, Mikael; Linse, Sara

    2010-01-01

    Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pKa values. However, pKa values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pKa values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pKa values were determined for carboxyl groups by monitoring their pH-dependent 13C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pKa values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pKa values of the folded and unfolded states and compared to experimental stability data. The use of experimental pKa values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone. PMID:21081085

  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. pKa Values for Side-Chain Carboxyl Groups of a PGB1 Variant Explain Salt and pH-Dependent Stability

    PubMed Central

    Lindman, Stina; Linse, Sara; Mulder, Frans A. A.; André, Ingemar

    2007-01-01

    Determination of pKa values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pKa values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using 1H-13C heteronuclear NMR in a structural context. The pKa values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pKa values can explain the pH dependence of protein stability but require pKa shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pKa value. We show also that 13C backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites. PMID:17040982

  14. Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

    PubMed

    Casarotto, M G; Basran, J; Badii, R; Sze, K H; Roberts, G C

    1999-06-22

    The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process. PMID:10387048

  15. Anchoring of both PKA-RIIα and 14-3-3θ regulates retinoic acid induced 16 mediated phosphorylation of heat shock protein 70

    PubMed Central

    Tang, Hai-Lin; Zhu, Shi-Ying; Zhao, Lan-Juan; Ren, Hao; Zhao, Ping; Qi, Zhong-Tian; Wang, Wen

    2015-01-01

    Our previous study reported that retinoic acid induced 16 (RAI16) could enhance tumorigenesis in hepatocellular carcinoma (HCC). However, the cellular functions of RAI16 are still unclear. In this study, by immunoprecipitation and tandem (MS/MS) mass spectrometry analysis, we identified that RAI16 interacted with the type II regulatory subunit of PKA (PKA-RIIα), acting as a novel protein kinase A anchoring protein (AKAP). In addition, RAI16 also interacted with heat shock protein 70 (HSP70) and 14-3-3θ. Further studies indicated that RAI16 mediated PKA phosphorylation of HSP70 at serine 486, resulting in anti-apoptosis events. RAI16 was also phosphorylated by the anchored PKA at serine 325, which promoted the recruitment of 14-3-3θ, which, in turn, inhibited RAI16 mediated PKA phosphorylation of HSP70. These findings offer mechanism insight into RAI16 mediated anti-apoptosis signaling in HCC. PMID:25900241

  16. Differences in Lysine pKa Values May Be Used to Improve NMR Signal Dispersion in Reductively Methylated Proteins

    PubMed Central

    Abraham, Sherwin J.; Kobayashi, Tomoyoshi; Solaro, R. John; Gaponenko, Vadim

    2009-01-01

    Summary Reductive methylation of lysine residues in proteins offers a way to introduce 13C methyl groups into otherwise unlabeled molecules. The 13C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of 13C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca2+-bound Calmodulin, Lysozyme, Ca2+-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of 13C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment. PMID:19280122

  17. Carboxyl pKa Values, Ion Pairs, Hydrogen Bonding, and the pH Dependence of Folding the Hyperthermophile Proteins Sac7d and Sso7d

    PubMed Central

    Clark, Andrew T.; Smith, Kelley; Muhandiram, Ranjith; Edmondson, Stephen P.; Shriver, John W.

    2007-01-01

    Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side chains in defining the stability and function of these Archaeal chromatin proteins, pKas were measured for all of the acidic residues in both proteins using 13C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low salt conditions. Two aspartate residues in Sso7d (D16 and D35) and a single glutamate residue (G54) showed significantly perturbed pKa values in low salt, indicating that the observed pH dependence of stability was primarily due to these three residues. The pH dependence of backbone amide NMR resonances demonstrated that perturbation of all three pKas was primarily the result of side chain-backbone amide hydrogen bonds. Titration data at higher salt for both Sso7d and Sac7d were consistent with this interpretation. Few of the significantly perturbed acidic pKas in Sac7d and Sso7d could be attributed to primarily ion pair or electrostatic interactions. A smaller perturbation of E48 (E47 in Sac7d) was ascribed to an ion pair interaction that may be important in defining the DNA binding surface. The small number (3) of significantly altered pKa values was in good agreement with a linkage analysis of the temperature, pH, and salt dependence of folding. The linkage of the ionization of two or more side chains to protein folding leads to apparent cooperativity in the pH dependence of folding, although each group titrates independently with a Hill coefficient near unity. These results demonstrate that the acid pH dependence of protein stability in these hyperthermophile proteins is due to independent titration of acidic residues with pKas perturbed primarily by hydrogen bonding of the side chain to the backbone. This work demonstrates the need for caution in using structural data alone to argue the importance of ion pairs in stabilizing

  18. Retinoic acid impairs estrogen signaling in breast cancer cells by interfering with activation of LSD1 via PKA.

    PubMed

    Ombra, Maria Neve; Di Santi, Annalisa; Abbondanza, Ciro; Migliaccio, Antimo; Avvedimento, Enrico Vittorio; Perillo, Bruno

    2013-05-01

    More than 70% of breast cancers in women require estrogens for cell proliferation and survival. 17β-estradiol (E2) effect on mammary target cells is almost exclusively mediated by its binding to the estrogen receptor-α (ERα) that joins chromatin where it assembles active transcription complexes. The proliferative and pro-survival action of estrogens is antagonized in most cases by retinoic acid (RA), even though the cognate retinoic acid receptor-α (RARα) cooperates with ERα on promoters of estrogen-responsive genes. We have examined at the molecular level the crosstalk between these nuclear receptors from the point of view of their control of cell growth and show here that RA reverts estrogen-stimulated transcription of the pivotal anti-apoptotic bcl-2 gene by preventing demethylation of dimethyl lysine 9 in histone H3 (HeK9me2). As we previously reported, this is obtained by means of E2-triggered activation of the lysine-specific demethylase 1 (LSD1), an enzyme that manages chromatin plasticity in order to allow specific movements of chromosomal regions within the nucleus. We find that E2 fuels LSD1 by inducing migration of the catalytic subunit of protein kinase A (PKA) into the nucleus, where it targets estrogen-responsive loci. RA rescues LSD1-dependent disappearance of H3K9me2 at bcl-2 regulatory regions upon the prevention of PKA assembly to the same sites. PMID:23507259

  19. Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pKa values by MALDI-TOF mass spectrometry

    PubMed Central

    Nelson, Kimberly J.; Day, Amanda E.; Zeng, Bubing B.; King, S. Bruce; Poole, Leslie B.

    2008-01-01

    Cysteine reactivity in enzymes is imparted to a large extent by the stabilization of the deprotonated form of the reduced cysteine (i.e. the thiolate) within the active site. While this is likely to be an important chemical attribute of many thiol-based enzymes including cysteine-dependent peroxidases (peroxiredoxins) and proteases, only relatively few pKa values have been determined experimentally. Presented here is a new technique for determining the pKa value of cysteine residues through quantitative mass spectrometry following chemical modification with an iodoacetamide-based reagent over a range of pH buffers. This isotope-coded reagent, N-phenyl iodoacetamide (iodoacetanilide), is readily prepared in deuterated (d5) and protiated (d0) versions and is more reactive toward free cysteine than is iodoacetamide. Using this approach, the pKa values for the two cysteine residues in Escherichia coli thioredoxin were determined to be 6.5 and > 10, in good agreement with previous reports using chemical modification approaches. This technique allows the pKa of specific cysteine residues to be determined in a clear, fast, and simple manner and, because cysteine residues on separate tryptic peptides are measured separately, is not complicated by the presence of multiple cysteines within the protein of interest. PMID:18162165

  20. 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...

  1. [Quantitative structure activity relationship models based on heuristic method and gene expression programming for the prediction of the pK(a) values of sulfa drugs].

    PubMed

    Li, Yu-qin; Si, Hong-zong; Xiao, Yu-liang; Liu, Cai-hong; Xia, Cheng-cai; Li, Ke; Qi, Yong-xiu

    2009-05-01

    Quantitative structure-property relationships (QSPR) were developed to predict the pK(a) values of sulfa drugs via heuristic method (HM) and gene expression programming (GEP). The descriptors of 31 sulfa drugs were calculated by the software CODESSA, which can calculate constitutional, topological, geometrical, electrostatic, and quantum chemical descriptors. HM was also used for the preselection of 4 appropriate molecular descriptors. Linear and nonlinear QSPR models were developed based on the HM and GEP separately and two prediction models lead to a good correlation coefficient (R) of 0.90 and 0.95. The two QSPR models are tseful in predicting pK(a) during the discovery of new drugs and providing theory information for studying the new drugs. PMID:19618723

  2. 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.

  3. 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

  4. 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

  5. pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

    PubMed Central

    Yuan, Shuguang; Le Roy, Katrien; Venken, Tom; Lammens, Willem; Van den Ende, Wim; De Maeyer, Marc

    2012-01-01

    Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst. PMID:22662155

  6. Predicting hydrogen-bond strengths from acid-base molecular properties. The pK(a) slide rule: toward the solution of a long-lasting problem.

    PubMed

    Gilli, Paola; Pretto, Loretta; Bertolasi, Valerio; Gilli, Gastone

    2009-01-20

    Unlike normal chemical bonds, hydrogen bonds (H-bonds) characteristically feature binding energies and contact distances that do not simply depend on the donor (D) and acceptor (:A) nature. Instead, their chemical context can lead to large variations even for a same donor-acceptor couple. As a striking example, the weak HO-H...OH(2) bond in neutral water changes, in acidic or basic medium, to the 6-fold stronger and 15% shorter [H(2)O...H...OH(2)](+) or [HO...H...OH](-) bonds. This surprising behavior, sometimes called the H-bond puzzle, practically prevents prediction of H-bond strengths from the properties of the interacting molecules. Explaining this puzzle has been the main research interest of our laboratory in the last 20 years. Our first contribution was the proposal of RAHB (resonance-assisted H-bond), a new type of strong H-bond where donor and acceptor are linked by a short pi-conjugated fragment. The RAHB discovery prompted new studies on strong H-bonds, finally leading to a general H-bond classification in six classes, called the six chemical leitmotifs, four of which include all known types of strong bonds. These studies attested to the covalent nature of the strong H-bond showing, by a formal valence-bond treatment, that weak H-bonds are basically electrostatic while stronger ones are mixtures of electrostatic and covalent contributions. The covalent component gradually increases as the difference of donor-acceptor proton affinities, DeltaPA, or acidic constants, DeltapK(a), approaches zero. At this limit, the strong and symmetrical D...H...A bonds formed can be viewed as true three-center-four-electron covalent bonds. These results emphasize the role PA/pK(a) equalization plays in strengthening the H-bond, a hypothesis often invoked in the past but never fully verified. In this Account, this hypothesis is reconsidered by using a new instrument, the pK(a) slide rule, a bar chart that reports in separate scales the pK(a)'s of the D-H proton donors and

  7. Highly Conserved Histidine Plays a Dual Catalytic Role in Protein Splicing: a pKa Shift Mechanism

    PubMed Central

    Du, Zhenming; Shemella, Philip T.; Liu, Yangzhong; McCallum, Scott A.; Pereira, Brian; Nayak, Saroj K.; Belfort, Georges; Belfort, Marlene; Wang, Chunyu

    2009-01-01

    Protein splicing is a precise auto-catalytic process in which an intein excises itself from a precursor with the concomitant ligation of the flanking sequences. Protein splicing occurs through acid-base catalysis in which the ionization states of active site residues are crucial to the reaction mechanism. In inteins, several conserved histidines have been shown to play important roles in protein splicing, including the most conserved “B-block” histidine. In this study, we have combined NMR pKa determination with quantum mechanics/molecular mechanics (QM/MM) modeling to study engineered inteins from Mycobacterium tuberculosis (Mtu) RecA intein. We demonstrate a dramatic pKa shift for the invariant B-block histidine, the most conserved residue among inteins. The B-block histidine has a pKa of 7.3 ± 0.6 in a precursor and a pKa of < 3.5 in a spliced intein. The pKa values and QM/MM data suggest that the B-block histidine has a dual role in the acid-base catalysis of protein splicing. This histidine likely acts as a general base to initiate splicing with an acyl shift and then as a general acid to cause the breakdown of the scissile bond. The proposed pKa shift mechanism accounts for the biochemical data supporting the essential role for the B-block histidine and for the absolute sequence conservation of this residue. PMID:19630416

  8. A fluorine scan at the catalytic center of thrombin: C--F, C--OH, and C--OMe bioisosterism and fluorine effects on pKa and log D values.

    PubMed

    Schweizer, Eliane; Hoffmann-Röder, Anja; Schärer, Kaspar; Olsen, Jacob A; Fäh, Christoph; Seiler, Paul; Obst-Sander, Ulrike; Wagner, Björn; Kansy, Manfred; Diederich, François

    2006-06-01

    A series of 16 tricyclic thrombin inhibitors was prepared by using the 1,3-dipolar cycloaddition of azomethine ylides derived from 3- or 4-hydroxyproline and 4-bromobenzaldehyde, with N-(4-fluorobenzyl)maleimide as the key step. The terminal pyrrolidine ring of the inhibitors was systematically substituted to explore the potential bioisosteric behavior of C-F, C-OH, and C-OMe residues pointing into the environment of the catalytic center of a serine protease. X-ray crystal structure analyses revealed a distinct puckering preference of this ring. Substitution by F, HO, and MeO has a strong effect on the basicity of the adjacent pyrrolidine nitrogen center which originates from two sigma-inductive pathways between this center and the electronegative O and F atoms. gem-Difluorination decreases the pKa value of this tertiary amine center to <2, making the conjugated ammonium ion a moderately strong acid. Unexpectedly, F substitution next to the nitrogen center reduced the lipophilicity of the ligands, as revealed by measurements of the logarithmic partition coefficient log D. The biological assays showed that all compounds are thrombin inhibitors with activities between Ki=0.08 and 2.17 microM. Bioisosteric behavior of F, HO, and MeO substituents was observed. Their electronegative F and O atoms undergo energetically similar polar interactions with positively polarized centers, such as the N atom of His 57 which is hydrogen bonded to the catalytic Ser 195. However, for energetically similar polar interactions of C-F, C-OH, and C-OMe to occur, sufficient space is necessary for the accommodation of the Me group of the C-OMe residue, and a H-bond acceptor must be present to prevent unfavorable desolvation of the C-OH residue. PMID:16892401

  9. cIEF for rapid pKa determination of small molecules: a proof of concept.

    PubMed

    Romand, Stéphanie; Schappler, Julie; Veuthey, Jean-Luc; Carrupt, Pierre-Alain; Martel, Sophie

    2014-10-15

    A capillary isoelectric focusing (cIEF) method was developed for the determination of the ionization constants (pKa) of small molecules. Two approaches used to decrease the electroosmotic flow (EOF) were compared: (i) a hydroxypropylcellulose (HPC) coated capillary in aqueous medium and (ii) the addition of glycerol to act as a viscosifying agent. The cIEF method with the glycerol medium was selected, and the ionization constants of 22 basic and 21 acidic compounds, including 15 pharmaceutical drugs, were determined, resulting in pKa values from 3.5 to 7.4 and 6.4 to 9.3, respectively. cIEF offers an interesting alternative to other techniques for pKa determination with low sample consumption, high throughput and low cost. PMID:24995703

  10. Spectrophotometric pKa determination of ionizable pharmaceuticals: Resolution of molecules with weak pH-dependent spectral shift.

    PubMed

    Dohoda, Deren; Tsinman, Konstantin; Tsinman, Oksana; Wang, Haotian; Tam, Kin Y

    2015-10-10

    The extent of ionization of a drug molecule at different pH values can be characterized by its pKa (acid dissociation constants). It is an important parameter in pharmaceutical development to rationalize the physiochemical and biopharmaceutical properties of the drug molecule. UV titration for pKa determination is one of the popular methods. The success of this method requires the molecule exhibiting strong pH-dependent spectral shift related to the ionization process. Depending on the proximity between the ionizable group and the chromophore, the spectral shift may not be strong enough to warrant a successful determination. In a previous study, it has been reported that a distance of three σ bonds between the chromophore and the ionizable group was the limit for a precise pKa determination. In this work, a UV titration method for pKa determination, with a particular emphasis on molecules with weak pH-dependent spectral shift is investigated. It has been shown that the pKa values determined from this study are in good agreement with those determined using potentiometric method and literature data (R(2)=0.998). Our methodology revealed that successful pKa determination is feasible even with a separation distance of five σ bonds between the chromophore and the ionizable group. PMID:26026267

  11. 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

  12. 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.

  13. 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

  14. Determination of pKa of felodipine using UV-Visible spectroscopy

    NASA Astrophysics Data System (ADS)

    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.

  15. Saccharomyces cerevisiae pyruvate kinase Pyk1 is PKA phosphorylation substrate in vitro.

    PubMed

    Cytryńska, M; Frajnt, M; Jakubowicz, T

    2001-09-25

    Fractionation of Saccharomyces cerevisiae postribosomal extract on DEAE-cellulose revealed two fractions of cAMP-dependent protein kinase (PKA-1 and PKA-2). The presence of PKA in both fractions was confirmed by immunoblotting with anti-Bcy1 antibodies. Yeast pyruvate kinase Pyk1 identified by amino acid microsequencing analysis and immunoblotting with anti-Pyk1 antibodies copurified with the PKA-1 but not the -2 fraction. Pyk1 can be phosphorylated by yeast PKA in vitro in the presence of cAMP and cGMP. Two-dimensional gel electrophoretic analysis revealed four phosphorylated forms of Pyk1 modified by PKA. In phosphorylation of Pyk1 mainly the Tpk2 catalytic subunit of yeast PKA was involved. PMID:11583852

  16. 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.

  17. Proteus mirabilis urease. Partial purification and inhibition by boric acid and boronic acids.

    PubMed

    Breitenbach, J M; Hausinger, R P

    1988-03-15

    Urease was purified 800-fold and partially characterized from Proteus mirabilis, the predominant microorganism associated with urinary stones. Boric acid is a rapid reversible competitive inhibitor of urease. The pH-dependence of inhibition exhibited pKa values of 6.25 and 9.3, where the latter value is probably due to the inherent pKa of boric acid. Three boronic acids also were shown to inhibit urease competitively. PMID:3291857

  18. Prediction of the pKa's of aqueous metal ion +2 complexes.

    PubMed

    Jackson, Virgil E; Felmy, Andrew R; Dixon, David A

    2015-03-26

    Aqueous metal ions play an important role in many areas of chemistry. The acidities of [Be(H2O)4](2+), [M(H2O)6](2+), M = Mg(2+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), and Hg(2+), and [M(H2O)n](2+), M = Ca(2+) and Sr(2+), n = 7 and 8, complexes have been predicted using density functional theory, second-order Møller-Plesset perturbation theory (MP2), and coupled cluster CCSD(T) theory in the gas phase. pKa's in aqueous solution were predicted by using self-consistent reaction field (SCRF) calculations with different solvation models. The most common binding motif of the majority of the metal +2 complexes is coordination number (CN) 6, with each hexaaquo cluster having reasonably high symmetry for the best arrangement of the water molecules in the first solvation shell. Be(2+) is tetracoordinated, but a second solvation shell of 8 waters is needed to predict the pKa. The Ca(2+) and Sr(2+) aquo clusters have a coordination number of 7 or 8 as found in terms of the energy of the reaction M(H2O)7(2+) + H2O → M(H2O)8(2+) and the pKa values. The calculated geometries are in reasonable agreement with experiment. The SCRF calculations with the conductor-like screening model (COSMO), and the conductor polarized continuum model (CPCM) using COSMO-RS radii, consistently agree best with experiment at the MP2/aug-cc-pVDZ and CCSD(T)/aug-cc-pVDZ levels of theory. The CCSD(T) level provides the most accurate pKa's, and the MP2 level also provides reliable predictions. Our predictions were used to elucidate the properties of metal +2 ion complexes. The pKa predictions provide confirmation of the size of the first solvation shell sizes. The calculations show that it is still difficult to predict pKa's using this cluster/implicit solvent approach to better than 1 pKa unit. PMID:25721568

  19. Direct NMR observation and pKa determination of the Asp102 side chain in a serine protease.

    PubMed

    Everill, Paul; Sudmeier, James L; Bachovchin, William W

    2012-02-01

    The pK(a) value of aspartic acid in the catalytic triad of serine proteases has been a pivotal element in essentially every mechanism proposed for these enzymes over the past 40 years, but has, until now, eluded direct determination. Here, we have used the multinuclear 3D-NMR pulse programs HCACO and HCCH-TOCSY to directly identify and study the side-chain resonances of the aspartate and glutamate residues in uniformly (13)C-labeled α-lytic protease. Resonances from four of the six residues were detected and assigned, including that of Asp(102), which is notably the weakest of the four. pH titrations have shown all of the carboxylate (13)C signals to have unusually low pK(a) values: 2.0, 3.2, and 1.7 for Glu(129), Glu(174), and Glu(229), respectively, and an upper limit of 1.5 for Asp(102). The multiple H-bonds to Asp(102), long known from X-ray crystal studies, probably account for its unusually low pK(a) value through preferential stabilization of its anionic form. These H-bonds probably also contribute to the weakness of the NMR resonances of Asp(102) by restricting its mobility. The Asp(102)(13)C(γ) atom responds to the ionization of His(57) in the resting enzyme and to the inhibitor-derived oxyanion in a chloromethyl ketone complex, observations that strongly support the assignment. The low pK(a) value of Asp(102) would appear to be incompatible with mechanisms involving strong Asp(102)-His(57) H-bonds or high pK(a) values, but is compatible with mechanisms involving normal Asp(102)-His(57) H-bonds and moving His(57) imidazole rings, such as the reaction-driven ring flip. PMID:22229736

  20. Biorelevant pK(a) (37 °C) predicted from the 2D structure of the molecule and its pK(a) at 25 °C.

    PubMed

    Sun, Na; Avdeef, Alex

    2011-09-10

    Values of the ionization constants at 37 °C, which are scarcely reported, are more meaningful for interpreting mechanisms of cellular transport by ionizable molecules and in mechanistic dissolution studies, which are often performed at the biorelevant temperature. An equation was developed where the pK(a) values of drug-like molecules determined at 25 °C can be simply converted to values at 37 °C, without additional measurement. The differences between the values, ΔpK(a)=pK(a)³⁷-pK(a)²⁵, were linearly fitted to a function of pK(a)²⁵ and the standard entropy of ionization, ΔS°, where the latter term was approximated by the five Abraham linear free energy solvation descriptors using multiple linear regression. The Abraham descriptors (H-bond donor and acceptor strengths, dipolar solute-solvent interactions potential, the pi- and n-electrons dispersion force, and molar volume) were determined from the 2-dimensional structure of the molecules. A total of 143 mostly drug-like molecules (207 pK(a) values at 25 °C and at 37 °C) were chosen for the study. The pK(a) values of many were determined here for the first time. Included were 34 weak acids, 85 weak bases, and 24 amphoteric compounds (6 ordinary ampholytes, 18 zwitterions). PMID:21652160

  1. 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

  2. General Analytical Procedure for Determination of Acidity Parameters of Weak Acids and Bases

    PubMed Central

    Pilarski, Bogusław; Kaliszan, Roman; Wyrzykowski, Dariusz; Młodzianowski, Janusz; Balińska, Agata

    2015-01-01

    The paper presents a new convenient, inexpensive, and reagent-saving general methodology for the determination of pKa values for components of the mixture of diverse chemical classes weak organic acids and bases in water solution, without the need to separate individual analytes. The data obtained from simple pH-metric microtitrations are numerically processed into reliable pKa values for each component of the mixture. Excellent agreement has been obtained between the determined pKa values and the reference literature data for compounds studied. PMID:25692072

  3. Using membrane composition to fine-tune the pKa of an optical liposome pH sensor†

    PubMed Central

    Clear, Kasey J.; Virga, Katelyn; Gray, Lawrence

    2016-01-01

    Liposomes containing membrane-anchored pH-sensitive optical probes are valuable sensors for monitoring pH in various biomedical samples. The dynamic range of the sensor is maximized when the probe pKa is close to the expected sample pH. While some biomedical samples are close to neutral pH there are several circumstances where the pH is 1 or 2 units lower. Thus, there is a need to fine-tune the probe pKa in a predictable way. This investigation examined two lipid-conjugated optical probes, each with appended deep-red cyanine dyes containing indoline nitrogen atoms that are protonated in acid. The presence of anionic phospholipids in the liposomes stabilized the protonated probes and increased the probe pKa values by < 1 unit. The results show that rational modification of the membrane composition is a general non-covalent way to fine-tune the pKa of an optical liposome sensor for optimal pH sensing performance. PMID:27087967

  4. Predicting Extreme pKa Shifts in Staphylococcal Nuclease Mutants with Constant pH Molecular Dynamics

    PubMed Central

    Arthur, Evan J.; Yesselman, Joseph D.; Brooks, Charles L.

    2011-01-01

    Accurate computational methods of determining protein and nucleic acid pKa values are vital to understanding pH-dependent processes in biological systems. In this paper we use the recently developed method constant pH molecular dynamics (CPHMD) to explore the calculation of highly-perturbed pKa values in variants of staphylococcal nuclease (SNase). Simulations were performed using the replica exchange (REX) protocol for improved conformational sampling with eight temperature windows, and yielded converged proton populations in a total sampling time of 4 ns. Our REX-CPHMD simulations resulted in calculated pKa values with an average unsigned error (AUE) of 0.75 pK units for the acidic residues in Δ+PHS, a hyperstable variant of SNase. For highly pKa-perturbed SNase mutants with known crystal structures, our calculations yielded an AUE of 1.5 pK units and for those mutants based on modeled structures an AUE of 1.4 pK units was found. Although a systematic underestimate of pK shifts was observed in most of the cases for the highly perturbed pK mutants, correlations between conformational rearrangement and plasticity associated with the mutation and error in pKa prediction was not evident in the data. This study further extends the scope of electrostatic environments explored using the REX-CPHMD methodology and suggests it is a reliable tool for rapidly characterizing ionizable amino acids within proteins even when modeled structures are employed. PMID:22002886

  5. Determination of Acid Dissociation Constants (pKa) of Bicyclic Thiohydantoin-Pyrrolidine Compounds in 20% Ethanol-Water Hydroorganic Solvent

    PubMed Central

    Nural, Yahya; Döndaş, H. Ali; Sarı, Hayati; Atabey, Hasan; Belveren, Samet; Gemili, Müge

    2014-01-01

    The acid dissociation constants of potential bioactive fused ring thiohydantoin-pyrrolidine compounds were determined by potentiometric titration in 20% (v/v) ethanol-water mixed at 25 ± 0.1°C, at an ionic background of 0.1 mol/L of NaCl using the HYPERQUAD computer program. Proton affinities of potential donor atoms of the ligands were calculated by AM1 and PM3 semiempiric methods. We found, potentiometrically, three different acid dissociation constants for 1a–f. We suggest that these acid dissociation constants are related to the carboxyl, enol, and amino groups. PMID:24799905

  6. Calculation of pK(a) in proteins with the microenvironment modulated-screened coulomb potential.

    PubMed

    Shan, Jufang; Mehler, Ernest L

    2011-12-01

    The MM-SCP has been applied to predict pK(a) values of titratable residues in wild type and mutants of staphylococcal nuclease (SNase). The calculations were based on crystal structures made available by the Garcia-Moreno Laboratory. In the mutants, mostly deeply buried hydrophobic residues were replaced with ionizable residues, and thus their pK(a) values could be measured and calculated using various methods. The data set used here consisted of a set of WT SNase for which His pK(a) at several ionic strengths had been measured, a set of mutants for which measured pK(a) were available and a set of 11 mutants for which the measured pK(a) were not known at the time of calculation. For this latter set, blind predictions were submitted to the protein pK(a) cooperative, 2009 workshop at Telluride, where the results of the blind predictions were discussed (the RMSD of the submitted set was 1.10 pH units). The calculations on the structures with known pK(a) indicated that in addition to weaknesses of the method, structural issues were observed that led to larger errors (>1) in pK(a) predictions. For example, different crystallographic conditions or steric clashes can lead to differences in the local environment around the titratable residue, which can produce large differences in the calculated pK(a) . To gain further insight into the reliability of the MM-SCP, pK(a) of an extended set of 54 proteins belonging to several structural classes were carried out. Here some initial results from this study are reported to help place the SNase results in the appropriate context. PMID:21748803

  7. Aspartate Embedding Depth Affects pHLIP’s Insertion pKa

    PubMed Central

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

    2014-01-01

    We have used the pHLIP® (pH Low Insertion Peptide) peptide 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 pH is lowered, pHLIP inserts spontaneously across the membrane as a spanning α-helix. pHLIP insertion is reversible when 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 further examine this protonation mechanism, we created pHLIP sequence variants in which the position of the two spanning aspartates (D14, D25) was moved up or down in the sequence. We hypothesized that 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 see if arginine snorkeling modulates the insertion pKa by affecting aspartate depth. Our results indicate both types of mutations change the insertion pKa, supporting the idea that aspartate depth is a participating parameter in determining pH dependence. We also show that pHLIP’s resistance to aggregation can be altered with our mutations, identifying a new criterion for improving pHLIP performance in vivo when targeting acidic disease tissues such as cancer and inflammation. PMID:23721379

  8. 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

  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. Nutritional value of D-amino acids, D-peptides, and amino acid derivatives in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper describes a method for determining the nutritional value of D-amino acids, D-peptides, and amino acid derivatives using a growth assay in mice fed a synthetic all-amino acid diet. A large number of experiments were carried out in which a molar equivalent of the test compound replaced a n...

  11. 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

  12. 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

  13. CALCULATION OF pKa IN PROTEINS WITH THE MICROENVIRONMENT MODULATED-SCREENED COULOMB POTENTIAL (MM-SCP)

    PubMed Central

    Shan, Jufang; Mehler, Ernest L.

    2011-01-01

    The MM-SCP has been applied to predict pKa values of titratable residues in wild type and mutants of staphylococcal nuclease (SNase). The calculations were based on crystal structures made available by the Garcia-Moreno Laboratory. In the mutants, mostly deeply buried hydrophobic residues were replaced with ionizable residues, and thus their pKa values could be measured and calculated using various methods. The data set used here consisted of a set of WT SNase for which His pKa at several ionic strengths had been measured, a set of mutants for which measured pKa were available and a set of 11 mutants for which the measured pKa were not known at the time of calculation. For this latter set, blind predictions were submitted to the protein pKa cooperative, 2009 workshop at Telluride, where the results of the blind predictions were discussed (the RMSD of the submitted set was 1.10 pH units). The calculations on the structures with known pKa indicated that in addition to weaknesses of the method, structural issues were observed that led to larger errors (>1) in pKa predictions. For example, different crystallography conditions or steric clashes can lead to differences in the local environment around the titratable residue, which can produce large differences in the calculated pKa. To gain further insight into the reliability of the MM-SCP, pKa of an extended set of 54 proteins belonging to several structural classes were carried out. Here some initial results from this study are reported to help place the SNase results in the appropriate context. PMID:21748803

  14. 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

  15. 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

  16. 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

  17. Self-assembled monolayers of NH2-terminated thiolates: order, pKa, and specific adsorption.

    PubMed

    Marmisollé, Waldemar A; Capdevila, Daiana A; de la Llave, Ezequiel; Williams, Federico J; Murgida, Daniel H

    2013-04-30

    Self-assembled monolayers (SAMs) of amino-terminated alkanethiols on Au were characterized by a combination of electrochemical (LSV, CV, and EIS) and spectroscopic (XPS and SER) techniques. Clear correlations were obtained between the apparent surface pKa values determined by impedimetric titrations and order parameters such as the content of trans conformers in the SAMs. These results contrast with previous studies that exhibit dispersions of up to 6 pH units in the reported pKa values. In addition, we determined that inorganic and organic phosphate species bind specifically to these SAMs mediating adsorption and heterogeneous electron transfer of positively charged macromolecules such as cytochrome c. PMID:23560885

  18. 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

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

    PubMed Central

    Chowdhury, Tahmeena; Köhler, Julia R.

    2015-01-01

    Summary 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. PMID:26173379

  20. Modelling, solubility and pK(a) of five sparingly soluble drugs.

    PubMed

    Domańska, Urszula; Pobudkowska, Aneta; Pelczarska, Aleksandra; Zukowski, Lukasz

    2011-01-17

    Drug solubility is an important aspect of drug development. The objective of this investigation was to measure solubilities of five drugs (cimetidine, phenylbutazone, fenbufen, nitrofurantoin, triamterene) at constant pH in range of temperature from 270 to 340K in three solvents: water, ethanol and 1-octanol with the dynamic-visual method and the saturation shake-flask method using spectrophotometric analysis. The Barton group contribution method was used for the calculations of molar volumes of solutes. The thermodynamic description of the solubility curves was made using the thermophysical properties obtained with the differential scanning microcalorimetry technique (DSC). The DSC measurements have shown different than existing in the literature enthalpies of melting for phenylbutazone and fenbufen. The experimental solubility data also differ from the literature data, normally measured at one, or two temperatures only. The solubility data have been correlated by means of three commonly known excess Gibbs energy, G(E) equations. The activity coefficients of drugs at saturated solutions were calculated from the experimental data. Reexamination of the pK(a) values using diluted solutions was made with the Bates-Schwarzenbach method for the pK(a) measurements. The association constants and corresponding pK(a) values of drugs were close to the most of the literature data. We hope that our new solubility data, thermophysical data, and pK(a) values will improve all prediction-methods and their precision. PMID:21034801

  1. EGFRvIII stimulates glioma growth and invasion through PKA-dependent serine phosphorylation of Dock180.

    PubMed

    Feng, H; Hu, B; Vuori, K; Sarkaria, J N; Furnari, F B; Cavenee, W K; Cheng, S-Y

    2014-05-01

    Glioblastomas (GBMs), the most common and malignant brain tumors, are highly resistant to current therapies. The failure of targeted therapies against aberrantly activated oncogenic signaling, such as that of the EGFR-PI3K/Akt pathway, underscores the urgent need to understand alternative downstream pathways and to identify new molecular targets for the development of more effective treatments for gliomas. Here, we report that EGFRvIII (ΔEGFR/de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in clinical GBM tumors, promotes glioma growth and invasion through protein kinase A (PKA)-dependent phosphorylation of Dock180, a bipartite guanine nucleotide exchange factor (GEF) for Rac1. We demonstrate that EGFRvIII induces serine phosphorylation of Dock180, stimulates Rac1 activation and glioma cell migration. Treatments of glioma cells using the PKA inhibitors H-89 and KT5720, overexpression of a PKA inhibitor (PKI), and in vitro PKA kinase assays show that EGFRvIII induction of serine phosphorylation of Dock180 is PKA-dependent. Significantly, PKA induces phosphorylation of Dock180 at amino acid residue S1250 that resides within its Rac1-activating DHR-2 domain. Expression of the Dock180(S1250L) mutant, but not wild type Dock180(WT), protein in EGFRvIII-expressing glioma cells inhibited receptor-stimulated cell proliferation, survival, migration in vitro and glioma tumor growth and invasion in vivo. Together, our findings describe a novel mechanism by which EGFRvIII drives glioma tumorigenesis and invasion through PKA-dependent phosphorylation of Dock180, thereby suggesting that targeting EGFRvIII-PKA-Dock180-Rac1 signaling axis could provide a novel pathway to develop potential therapeutic strategies for malignant gliomas. PMID:23728337

  2. 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). PMID:27058878

  3. 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.

  4. Density Functional Theory Calculation of pKa's of Thiols in Aqueous Solution Using Explicit Water Molecules and the Polarizable Continuum Model.

    PubMed

    Thapa, Bishnu; Schlegel, H Bernhard

    2016-07-21

    The pKa's of substituted thiols are important for understanding their properties and reactivities in applications in chemistry, biochemistry, and material chemistry. For a collection of 175 different density functionals and the SMD implicit solvation model, the average errors in the calculated pKa's of methanethiol and ethanethiol are almost 10 pKa units higher than for imidazole. A test set of 45 substituted thiols with pKa's ranging from 4 to 12 has been used to assess the performance of 8 functionals with 3 different basis sets. As expected, the basis set needs to include polarization functions on the hydrogens and diffuse functions on the heavy atoms. Solvent cavity scaling was ineffective in correcting the errors in the calculated pKa's. Inclusion of an explicit water molecule that is hydrogen bonded with the H of the thiol group (in neutral) or S(-) (in thiolates) lowers error by an average of 3.5 pKa units. With one explicit water and the SMD solvation model, pKa's calculated with the M06-2X, PBEPBE, BP86, and LC-BLYP functionals are found to deviate from the experimental values by about 1.5-2.0 pKa units whereas pKa's with the B3LYP, ωB97XD and PBEVWN5 functionals are still in error by more than 3 pKa units. The inclusion of three explicit water molecules lowers the calculated pKa further by about 4.5 pKa units. With the B3LYP and ωB97XD functionals, the calculated pKa's are within one unit of the experimental values whereas most other functionals used in this study underestimate the pKa's. This study shows that the ωB97XD functional with the 6-31+G(d,p) and 6-311++G(d,p) basis sets, and the SMD solvation model with three explicit water molecules hydrogen bonded to the sulfur produces the best result for the test set (average error -0.11 ± 0.50 and +0.15 ± 0.58, respectively). The B3LYP functional also performs well (average error -1.11 ± 0.82 and -0.78 ± 0.79, respectively). PMID:27327957

  5. Subcellular dynamics of type II PKA in neurons

    PubMed Central

    Zhong, Haining; Sia, Gek-Ming; Sato, Takashi R.; Gray, Noah W.; Mao, Tianyi; Khuchua, Zaza; Huganir, Richard L.; Svoboda, Karel

    2009-01-01

    SUMMARY Protein kinase A (PKA) plays multiple roles in neurons. The localization and specificity of PKA are largely controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA in neurons, and the roles of specific AKAPs, are poorly understood. We imaged the distribution of type II PKA in hippocampal and cortical layer 2/3 pyramidal neurons in vitro and in vivo. PKA was concentrated in dendritic shafts compared to the soma, axons and dendritic spines. This spatial distribution was imposed by the microtubule-binding protein MAP2, indicating that MAP2 is the dominant AKAP in neurons. Following cAMP elevation, catalytic subunits dissociated from the MAP2-tethered regulatory subunits and rapidly moved to become enriched in nearby spines. The spatial gradient of type II PKA between dendritic shafts and spines was critical for the regulation of synaptic strength and long-term potentiation. The localization and activity-dependent translocation of type II PKA are therefore important determinants of PKA function. PMID:19447092

  6. Signaling Diversity of PKA Achieved Via a Ca2+-cAMP-PKA Oscillatory Circuit

    PubMed Central

    Ni, Qiang; Ganesan, Ambhighainath; Aye-Han, Nwe-Nwe; Gao, Xinxin; Allen, Michael D.; Levchenko, Andre; Zhang, Jin

    2010-01-01

    Many protein kinases are key nodal signaling molecules that regulate a wide range of cellular functions. These functions may require complex spatiotemporal regulation of kinase activities. Here, we show that Protein Kinase A (PKA), Ca2+ and cAMP oscillate in sync in insulin-secreting MIN6 β cells, forming a highly integrated oscillatory circuit. We found that PKA activity was essential for this oscillatory circuit, and was capable of not only initiating the signaling oscillations but also modulating their frequency, thereby diversifying the spatiotemporal control of downstream signaling. Our findings suggest that exquisite temporal control of kinase activity, mediated via signaling circuits resulting from cross-regulation of signaling pathways, can encode diverse inputs into temporal parameters such as oscillation frequency, which in turn contributes to proper regulation of complex cellular functions in a context-dependent manner. PMID:21102470

  7. 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. PMID:27083264

  8. A buried lysine that titrates with a normal pKa: Role of conformational flexibility at the protein–water interface as a determinant of pKavalues

    PubMed Central

    Harms, Michael J.; Schlessman, Jamie L.; Chimenti, Michael S.; Sue, Gloria R.; Damjanović, Ana; García-Moreno E., Bertrand

    2008-01-01

    Previously we reported that Lys, Asp, and Glu residues at positions 66 and 92 in staphylococcal nuclease (SNase) titrate with pKa values shifted by up to 5 pKa units in the direction that promotes the neutral state. In contrast, the internal Lys-38 in SNase titrates with a normal pKa. The crystal structure of the L38K variant shows that the side chain of Lys-38 is buried. The ionizable moiety is ∼7 Å from solvent and ion paired with Glu-122. This suggests that the pKa value of Lys-38 is normal because the energetic penalty for dehydration is offset by a favorable Coulomb interaction. However, the pKa of Lys-38 was also normal when Glu-122 was replaced with Gln or with Ala. Continuum electrostatics calculations were unable to reproduce the pKa of Lys-38 unless the protein was treated with an artificially high dielectric constant, consistent with structural reorganization being responsible for the normal pKa value of Lys-38. This reorganization must be local because circular dichroism and NMR spectroscopy indicate that the L38K protein is native-like under all conditions studied. In molecular dynamics simulations, the ion pair between Lys-38 and Glu-122 is unstable. The simulations show that a minor rearrangement of a loop is sufficient to allow penetration of water to the amino moiety of Lys-38. This illustrates both the important roles of local flexibility and water penetration as determinants of pKa values of ionizable groups buried near the protein–water interface, and the challenges faced by structure-based pKa calculations in reproducing these effects. PMID:18369193

  9. 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...

  10. Estimation of Molecular Acidity via Electrostatic Potential at the Nucleus and Valence Natural Atomic Orbitals

    PubMed Central

    Liu, Shubin; Pedersen, Lee G.

    2009-01-01

    An effective approach of estimating molecular pKa values from simple density functional calculations is proposed in this work. Both the molecular electrostatic potential (MEP) at the nucleus of the acidic atom and the sum of valence natural atomic orbitals are employed for three categories of compounds, amines and anilines, carbonyl acids and alcohols, and sulfonic acids and thiols. A strong correlation between experimental pKa values and each of these two quantities for each of the three categories has been discovered. Moreover, if the MEP is subtracted by the isolated atomic MEP for each category of compounds, we observe a single unique linear relationship between the resultant MEP difference and experimental pKa data of amines, anilines, carbonyl acids, alcohols, sulfonic acids, thiols, and their substituents. These results can generally be utilized to simultaneously estimate pKa values at multiple sites with a single calculation for either relatively small molecules in drug design or amino acids in proteins and macromolecules. PMID:19317439

  11. Oil Biotechnology: Value-Added Products and Bioactive Fatty Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    During my 40+ years research career, I have been working on "biocatalysis" of hydrophobic organic compounds, both petroleum oil and vegetable oil, to convert them to value-added products. "Biocatalysis" is defined as the use of a biocatalyst such as whole microbial cells or enzymes, in an aqueous o...

  12. Value of furfural/ethanol coproduction from acid hydrolysis processes

    SciTech Connect

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

    1983-08-01

    The benefits of two modifications in the acid hydrolysis of cellulosic feedstocks for the production of ethanol fuels were investigated: marketing of the by-product furfural and xylose fermentation. Preliminary analysis indicate that the furfural by-product furfural and xylose fermentation. Perliminary analyses indicate that the furfural by-product credit is more beneficial at a minimum net profit of $0.08/lb of furfural. For this credit to have a major impact on ethanol production costs, new markets for large quantities of furfural must be identified. Furfural can be an alternative feedstock for hydrocarbon-based commodity chemicals. The costs and profitabilities of producing five chemicals from furfurals as opposed to conventional hydrocarbon-based feedstocks were studied. The furfural processes for production of styrene and butadiene were found to be marginally competitive or not competitive. The furfural processes for adipic acid, maleic anhydride, and butanol could be competitive. Results of analysis by a computer model of the petrochemical industry indicate that with furfural markets additional to these three furfural processes, over 2.5 x 10/sup 9/ gal of ethanol could be marketed at about $1.00/gal. Converting the xylose stream to ethanol has about the same effect on the selling price of ethanol as the furfural credit. The greatest ethanol production will result from xylose fermentation, but the furfural credit offers large near-term profits and has a more diversified impact on reducing petroleum product demand. 6 figures, 17 tables.

  13. Fmoc-diphenylalanine self-assembly mechanism induces apparent pKa shifts.

    PubMed

    Tang, Claire; Smith, Andrew M; Collins, Richard F; Ulijn, Rein V; Saiani, Alberto

    2009-08-18

    We report the effect of pH on the self-assembly process of Fmoc-diphenylalanine (Fmoc-FF) into fibrils consisting of antiparallel beta-sheets, and show that it results in two apparent pKa shifts of approximately 6.4 and approximately 2.2 pH units above the theoretical pKa (3.5). Using Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), wide angle X-ray scattering (WAXS), and oscillatory rheology, these two transitions were shown to coincide with significant structural changes. An entangled network of flexible fibrils forming a weak hydrogel dominates at high pH, while nongelling flat rigid ribbons form at intermediate pH values. Overall, this study provides further understanding of the self-assembly mechanism of aromatic short peptide derivatives. PMID:19537819

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

    PubMed Central

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

    1986-01-01

    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. PMID:3458179

  15. The Nature of Allosteric Inhibition in Glutamate Racemase: discovery and characterization of a cryptic inhibitory pocket using atomistic MD simulations and pKa calculations

    PubMed Central

    Whalen, Katie L.; Tussey, Kenneth B.; Blanke, Steven R.; Spies, M. Ashley

    2011-01-01

    Enzyme inhibition via allostery, in which the ligand binds remotely from the active site, is a poorly understood phenomenon, and represents a significant challenge to structure-based drug design. Dipicolinic acid (DPA), a major component of Bacillus spores, is shown to inhibit glutamate racemase from Bacillus anthracis, a monosubstrate/monoproduct enzyme, in a novel allosteric fashion. Glutamate racemase has long been considered an important drug target for its integral role in bacterial cell wall synthesis. The DPA binding mode was predicted via multiple docking studies and validated via site-directed mutagenesis at the binding locus, while the mechanism of inhibition was elucidated with a combination of Blue Native PAGE, molecular dynamics simulations, free energy and pKa calculations. Inhibition by DPA not only reveals a novel cryptic binding site, but also represents a form of allosteric regulation that exploits the interplay between enzyme conformational changes, fluctuations in the pKa values of buried residues and catalysis. The potential for future drug development is discussed. PMID:21395329

  16. [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

  17. 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

  18. Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements

    PubMed Central

    Mosley, Pamela L.; Daniels, Kyle G.; Oas, Terrence G.

    2015-01-01

    The pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-L-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ~0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function. PMID:26267651

  19. 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. PMID:22502616

  20. 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. PMID:25514239

  1. 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.

  2. 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

  3. 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

  4. 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

  5. The concerted action of a positive charge and hydrogen bonds dynamically regulates the pKa of the nucleophilic cysteine in the NrdH-redoxin family.

    PubMed

    Van Laer, Koen; Oliveira, Margarida; Wahni, Khadija; Messens, Joris

    2014-02-01

    NrdH-redoxins shuffle electrons from the NADPH pool in the cell to Class Ib ribonucleotide reductases, which in turn provide the precursors for DNA replication and repair. NrdH-redoxins have a CVQC active site motif and belong to the thioredoxin-fold protein family. As for other thioredoxin-fold proteins, the pK(a) of the nucleophilic cysteine of NrdH-redoxins is of particular interest since it affects the catalytic reaction rate of the enzymes. Recently, the pK(a) value of this cysteine in Corynebacterium glutamicum and Mycobacterium tuberculosis NrdH-redoxins were determined, but structural insights explaining the relatively low pK(a) remained elusive. We subjected C. glutamicum NrdH-redoxin to an extensive molecular dynamics simulation to expose the factors regulating the pK(a) of the nucleophilic cysteine. We found that the nucleophilic cysteine receives three hydrogen bonds from residues within the CVQC active site motif. Additionally, a fourth hydrogen bond with a lysine located N-terminal of the active site further lowers the cysteine pK(a). However, site-directed mutagenesis data show that the major contribution to the lowering of the cysteine pK(a) comes from the positive charge of the lysine and not from the additional Lys-Cys hydrogen bond. In 12% of the NrdH-redoxin family, this lysine is replaced by an arginine that also lowers the cysteine pK(a). All together, the four hydrogen bonds and the electrostatic effect of a lysine or an arginine located N-terminally of the active site dynamically regulate the pK(a) of the nucleophilic cysteine in NrdH-redoxins. PMID:24243781

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

    PubMed

    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

  7. 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

  8. 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.

  9. 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.

  10. 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. PMID:26996259

  11. Acid deposition and air quality related values in north central Colorado wilderness areas. Final report

    SciTech Connect

    Hidy, G.M.

    1995-05-01

    Terrestrial and aquatic ecosystem response to atmospheric acid, sulfur, and nitrate deposition has been studied only in a very limited way in Colorado wilderness areas. However, the observed deposition rates in north central Colorado remain low relative to affected areas in the eastern United States and well within a range where no adverse ecological effects are expected. This report presents a survey of scientific information describing acid deposition and air quality related values, which may have implications for utility plant operations.

  12. 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

  13. 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

  14. 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

  15. Amino acid composition and crude protein values of some Cyanobacteria from Çanakkale (Turkey).

    PubMed

    Akgül, Rıza; Kızılkaya, Bayram; Akgül, Füsun; Erduğan, Hüseyin

    2015-09-01

    Cyanobacteria (blue-green algae) form an important component of integrated nutrient managements in agriculture and are exploited in commercial biotechnological ventures. In this study, Rivularia bullata (Poir) Berkeley ex Bornet & Flahault, Nostocs pongiaeforme C. Agardh ex Bornet & Flahault were researched for their amino acid composition and crude protein values. R. bullata was collected from coastal zones of the Gulf of Saros and N. spongiaeforme from the Ayazma Stream. The levels of amino acids were measured in algae samples using EZ: fast kits (EZ: fast GC/FID Protein Hydrolysate Amino Acid Kit) by gas chromatography. The crude proteins of samples were determined by the Kjeldahl method and were calculated using a nitrogen conversion factor of 6.25. Thirty-two amino acids were investigated, for N. spongiaeforme eight free essential amino acids (EAA), eight free non-essential amino acids (NEAA) and eleven other amino acids (OAA); for R. bullata eight EAA, eight NEAA and eight OAA were detected. Aspartic acid is the major constituent for both species. The total protein percents were determined for N. spongiaeforme as % 19.83 and for R. bullata as % 6.15. When considering the increasing world population and reducing natural products; Cyanobacteria will benew feed sources for all living. PMID:26408895

  16. Preorganized Hydrogen Bond Donor Catalysts: Acidities and Reactivities.

    PubMed

    Samet, Masoud; Kass, Steven R

    2015-08-01

    Measured DMSO pKa values for a series of rigid tricyclic adamantane-like triols containing 0-3 trifluoromethyl groups (i.e., 3(0)-3(3)) are reported. The three compounds with CF3 substituents are similar or more acidic than acetic acid (pKa = 13.5 (3(1)), 9.5 (3(2)), 7.3 (3(3)) vs 12.6 (HOAc)), and the resulting hydrogen bond network enables a remote γ-trifluoromethyl group to enhance the acidity as well as one located at the α-position. Catalytic abilities of 3(0)-3(3) were also examined. In a nonpolar environment a rate enhancement of up to 100-fold over flexible acyclic analogs was observed presumably due to an entropic advantage of the locked-in structure. Gas-phase acidities are found to correlate with the catalytic activity better than DMSO pKa values and appear to be a better measure of acidities in low dielectric constant media. These trends are reduced or reversed in polar solvents highlighting the importance of the reaction environment. PMID:26140305

  17. The role of hydrophobic microenvironments in modulating pKa shifts in proteins.

    PubMed

    Mehler, E L; Fuxreiter, M; Simon, I; Garcia-Moreno, E B

    2002-08-01

    The screened Coulomb potential (SCP) method, combined with a quantitative description of the microenvironments around titratable groups, based on the Hydrophobic Fragmental Constants developed by Rekker, has been applied to calculate the pK(a) values of groups embedded in extremely hydrophobic microenvironments in proteins. This type of microenvironment is not common; but constitutes a small class, where the protein's architecture has evolved to lend special properties to the embedded residue. They are of significant interest because they are frequently important in catalysis and in proton and electron transfer reactions. In the SCP treatment these special cases are treated locally and therefore do not affect the accuracy of the pK(a) values calculated for other residues in less hydrophobic environments. Here the calibration of the algorithm is extended with the help of earlier results from lysozyme and of three mutants of staphylococcal nuclease (SNase) that were specially designed to measure the energetics of ionization of titratable groups buried in extremely hydrophobic microenvironments. The calibrated algorithm was subsequently applied to a fourth mutant of SNase and then to a very large dimeric amine oxidase of 1284 residues, where 334 are titratable. The observed pK(a) shifts of the buried residues are large (up to 4.7 pK units), and all cases are well reproduced by the calculations with a root mean square error of 0.22. These results support the hypothesis that protein electrostatics can only be described correctly and self-consistently if the inherent heterogeneity of these systems is properly accounted for. PMID:12112696

  18. 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

  19. Biodiesel production from high acid value waste frying oil catalyzed by superacid heteropolyacid.

    PubMed

    Cao, Fenghua; Chen, Yang; Zhai, Fengying; Li, Jing; Wang, Jianghua; Wang, Xiaohong; Wang, Shengtian; Zhu, Weimin

    2008-09-01

    Transesterification of waste cooking oil with high acid value and high water contents using heteropolyacid H3PW12O40 x 6H2O (PW12) as catalyst was investigated. The hexahydrate form of PW(12) was found to be the most promising catalyst which exhibited highest ester yield 87% for transesterification of waste cooking oil and ester yield 97% for esterification of long-chain palmitic acid, respectively. The PW12 acid catalyst shows higher activity under the optimized reaction conditions compared with conventional homogeneous catalyst sulfuric acid, and can easily be separated from the products by distillation of the excess methanol and can be reused more times. The most important feature of this catalyst is that the catalytic activity is not affected by the content of free fatty acids (FFAs) and the content of water in the waste cooking oil and the transesterification can occur at a lower temperature (65 degrees C), a lower methanol oil ratio (70:1) and be finished within a shorter time. The results illustrate that PW12 acid is an excellent water-tolerant and environmentally benign acid catalyst for production of biodiesel from waste cooking oil. PMID:18646228

  20. AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells.

    PubMed

    Hallows, Kenneth R; Alzamora, Rodrigo; Li, Hui; Gong, Fan; Smolak, Christy; Neumann, Dietbert; Pastor-Soler, Núria M

    2009-04-01

    Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK. PMID:19211918

  1. 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

  2. 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

  3. 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...

  4. Fermentation inhibitors and their recovery from acid wood hydrolyzates as chemicals of value

    SciTech Connect

    Tran, Hao Cao.

    1986-01-01

    The principal objective of this study was to demonstrate the technical feasibility of fermentation inhibition removal from acid hydrolyzates of lignocellulosic materials and recovery of the by-products of acid hydrolysis as chemicals of value. The acid hydrolyzates of mixed hardwoods were produced from the continuous flow demonstration unit (CFDU) at the University of California Forest Products Laboratory. Two separate liquid product streams were the prehydrolyzate (S1H) and the second stage hydrolyzate (S2H). Candida utilis NRRL Y-900 and Saccharomyces cerevisiae NRRL 2034 were used to monitor the fermentability of these two product streams, respectively. Two pretreatment methods were investigated in detail: flashing and solvent extraction. For the S1H, flashing was adequate to enable cell production with Candida utilis. The S2H rehired solvent extraction to ensure ethanolic fermentation at high concentrations of glucose (12% or higher). A solvent was selected for the extraction process and process flow designs were proposed to recycle the solvent and isolate commercial by-products, including furfural, methanol, acetic acid and, potentially, formic acid, levulinic acid and 5-hydroxymethylfurfural.

  5. 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. PMID:18546174

  6. 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. PMID:25668697

  7. 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.

  8. 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

  9. Single Turnover Autophosphorylation Cycle of the PKA RIIβ Holoenzyme.

    PubMed

    Zhang, Ping; Knape, Matthias J; Ahuja, Lalima G; Keshwani, Malik M; King, Charles C; Sastri, Mira; Herberg, Friedrich W; Taylor, Susan S

    2015-07-01

    To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme typically nucleates a macromolecular complex or a "PKA signalosome." Using the RIIβ holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIβ subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIβ holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIβ holoenzyme since the RIIβ holoenzyme is located close to ion channels. The 2.8Å structure of an RIIβp2:C2:(Ca2ADP)2 holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-(β,γ-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The holoenzyme serves as a "product trap" because of the slow off-rate of the pRIIβ subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIβ signaling cycle, we show that release of pRIIβ in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site) inhibits holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RIIβ holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II holoenzymes. PMID:26158466

  10. Challenges in pKa Predictions for Proteins: The case of Asp213 in Human Proteinase 3

    NASA Astrophysics Data System (ADS)

    Hajjar, Eric; Dejaegere, Annick; Reuter, Nathalie

    2009-09-01

    Knowledge of the protonation states of the ionizable residues in an enzyme is a prerequisite to an accurate description of its structure and mechanism. In practice, the use of the inappropriate protonation state for an amino acid in a molecular modeling computation (e.g., molecular dynamics simulation) is likely to lead to unrealistic results. Although methods using solvers of the linearized Poisson-Boltzmann equation have proven to yield accurate pKa predictions, they bear a number of limitations. They are quite demanding in terms of computational power and are sensitive to representation of the charges and their position (force field and protein conformation). Moreover they depend on the choice of a dielectric constant for the protein interior. In this manuscript, we describe the difficulties met when trying to predict the protonation state of a buried amino acid, located in a protein for which very little biochemical data is available. Such a case is highly representative of the challenges faced in theoretical biology studies. Proteinase 3 (PR3) is an enzyme involved in proteolytic events associated with inflammation. It is a potential target in the development of new anti-inflammatory therapeutic strategies. We report the results of pKa predictions of the aspartic acid 213 of PR3 with a FDPB solver. We probed the influence of the choice of the dielectric constant for the protein interior ɛp and the benefits of conformational sampling by molecular dynamics (MD) on the pKa prediction of this carboxylate group. Using only the FDPB calculations, we could not conclude on the protonation state of Asp213. MD simulations confronted to knowledge of the ligand-binding and reaction mechanism led us to decide on a protonated form of this aspartic acid. We also demonstrate that the use of the wrong protonation state leads to an unreliable structural model for PR3. pKa prediction with a fast empirical method yielded a pKa of 8.4 for Asp213, which is in agreement with our

  11. Microbial Transformation of Nitriles to High-Value Acids or Amides

    NASA Astrophysics Data System (ADS)

    Chen, Jing; Zheng, Ren-Chao; Zheng, Yu-Guo; Shen, Yin-Chu

    Biotransformation of nitriles mediated by nitrile-amide converting enzymes has attracted considerable attention and developed tremendously in the recent years in China since it offers a valuable alternative to traditional chemical reaction which requires harsh conditions. As a result, an upsurge of these promising enzymes (including nitrile hydratase, nitrilase and amidase) has been taking place. This review aims at describing these enzymes in detail. A variety of microorganisms harboring nitrile-amide converting activities have been isolated and identified in China, some of which have already applied with moderate success. Currently, a wide range of high-value compounds such as aliphatic, alicyclic, aromatic and heterocyclic amides and their corresponding acids were provided by these nitrile-amide degra-ding organisms. Simultaneously, with the increasing demand of chiral substances, the enantioselectivity of the nitrilase superfamily is widely investigated and exploited in China, especially the bioconversion of optically active α-substituted phenylacetamides, acids and 2,2-dimethylcyclopropanecarboxamide and 2,2-dimethylcyclopropanecarboxylic acid by means of the catalysts exhibiting excellent stereoselectivity. Besides their synthetic value, the nitrile-amide converting enzymes also play an important role in environmental protection. In this context, cloning of the genes and expression of these enzymes are presented. In the near future in China, an increasing number of novel nitrile-amide converting organisms will be screened and their potential in the synthesis of useful acids and amides will be further exploited.

  12. Isoform-specific targeting of PKA to multivesicular bodies

    PubMed Central

    Day, Michele E.; Gaietta, Guido M.; Sastri, Mira; Koller, Antonius; Mackey, Mason R.; Scott, John D.; Perkins, Guy A.; Ellisman, Mark H.

    2011-01-01

    Although RII protein kinase A (PKA) regulatory subunits are constitutively localized to discrete cellular compartments through binding to A-kinase–anchoring proteins (AKAPs), RI subunits are primarily diffuse in the cytoplasm. In this paper, we report a novel AKAP-dependent localization of RIα to distinct organelles, specifically, multivesicular bodies (MVBs). This localization depends on binding to AKAP11, which binds tightly to free RIα or RIα in complex with catalytic subunit (holoenzyme). However, recruitment to MVBs occurs only with the release of PKA catalytic subunit (PKAc). This recruitment is reversed by reassociation with PKAc, and it is disrupted by the presence of AKAP peptides, mutations in the RIα AKAP-binding site, or knockdown of AKAP11. Cyclic adenosine monophosphate binding not only unleashes active PKAc but also leads to the targeting of AKAP11:RIα to MVBs. Therefore, we show that the RIα holoenzyme is part of a signaling complex with AKAP11, in which AKAP11 may direct RIα functionality after disassociation from PKAc. This model defines a new paradigm for PKA signaling. PMID:21502359

  13. 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

  14. 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.

  15. Addressing the Glycine-Rich Loop of Protein Kinases by a Multi-Facetted Interaction Network: Inhibition of PKA and a PKB Mimic.

    PubMed

    Lauber, Birgit S; Hardegger, Leo A; Asraful, Alam K; Lund, Bjarte A; Dumele, Oliver; Harder, Michael; Kuhn, Bernd; Engh, Richard A; Diederich, François

    2016-01-01

    Protein kinases continue to be hot targets in drug discovery research, as they are involved in many essential cellular processes and their deregulation can lead to a variety of diseases. A series of 32 enantiomerically pure inhibitors was synthesized and tested towards protein kinase A (PKA) and protein kinase B mimic PKAB3 (PKA triple mutant). The ligands bind to the hinge region, ribose pocket, and glycine-rich loop at the ATP site. Biological assays showed high potency against PKA, with Ki values in the low nanomolar range. The investigation demonstrates the significance of targeting the often neglected glycine-rich loop for gaining high binding potency. X-ray co-crystal structures revealed a multi-facetted network of ligand-loop interactions for the tightest binders, involving orthogonal dipolar contacts, sulfur and other dispersive contacts, amide-π stacking, and H-bonding to organofluorine, besides efficient water replacement. The network was analyzed in a computational approach. PMID:26578105

  16. [Lipase-catalyzed production of biodiesel from high acid value waste oil with ultrasonic assistant].

    PubMed

    Wang, Jian-Xun; Huang, Qing-De; Huang, Feng-Hong; Wang, Jiang-Wei; Huang, Qin-Jie

    2007-11-01

    Biodiesel fuel produced with the enzyme-catalyzed esterification and transesterification of high acid value waste oil through ultrasonic assistant was explored. Propyl oleate, biodiesel, converted from high acid value waste oil and 1-proponal catalyzed with immobilized lipases from Candida antarctica and Aspergillus oryzae in conditions of ultrasonic assistant. Commercial immobilized lipase Novozym 435 from C. antarctica was used as biocatalyst catalyzing high acid value waste oil and 1-proponal esterification and transesterification to propyl oleate under the ultrasonic assistant conditions and different conditions such as lipases amounts, initiatory molar ratio of propanol to oil, frequency of ultrasonic and power of ultrasonic were investigated and optimized. It is revealed that the enzymatic activity of Novozym435 is enhanced and, in particular, enzyme-catalyzed transesterification activity is enhanced obviously under the ultrasonic assistant conditions. Low frequency and mild energy ultrasonic is a key factor for enhancing enzymatic activity, emulsifying oil-propanol system and accelerating the speed of produce diffusing in the system. Under the optimal ultrasonic assistant reaction conditions, such as Novozym435 amounts 8% by oil quantity, initiatory molar ratio of propanol to oil 3:1, frequency of ultrasonic 28 KHz, power of ultrasonic 100 W and temperature of water batch 40-45 degrees C, the conversion ratio to propyl oleate reached to 94.86% in 50 mins in comparison with the highest conversion ratio to propyl oleate 84.43% under the conventional mechanical agitation conditions. Furthermore, it is demonstrated that various short chain linear and branched alcohols (C1-C5) show high conversion ratio to fatty acid alkyl esters (biodiesel) under the optimal ultrasonic assistant reaction conditions. On the other hand, ultrasonic energy is propitious to reduce the adsorption of product propyl oleate, by-product glycerol and other emplastics in system on the

  17. 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).

  18. Predictive Value of 8 Genetic Loci for Serum Uric Acid Concentration

    PubMed Central

    Gunjača, Grgo; Boban, Mladen; Pehlić, Marina; Zemunik, Tatijana; Budimir, Danijela; Kolčić, Ivana; Lauc, Gordan; Rudan, Igor; Polašek, Ozren

    2010-01-01

    Aim To investigate the value of genomic information in prediction of individual serum uric acid concentrations. Methods Three population samples were investigated: from isolated Adriatic island communities of Vis (n = 980) and Korčula (n = 944), and from general population of the city of Split (n = 507). Serum uric acid concentration was correlated with the genetic risk score based on 8 previously described genes: PDZK1, GCKR, SLC2A9, ABCG2, LRRC16A, SLC17A3, SLC16A9, and SLC22A12, represented by a total of 16 single-nucleotide polymorphisms (SNP). The data were analyzed using classification and regression tree (CART) and general linear modeling. Results The most important variables for uric acid prediction with CART were genetic risk score in men and age in women. The percent of variance for any single SNP in predicting serum uric acid concentration varied from 0.0%-2.0%. The use of genetic risk score explained 0.1%-2.5% of uric acid variance in men and 3.9%-4.9% in women. The highest percent of variance was obtained when age, sex, and genetic risk score were used as predictors, with a total of 30.9% of variance in pooled analysis. Conclusion Despite overall low percent of explained variance, uric acid seems to be among the most predictive human quantitative traits based on the currently available SNP information. The use of genetic risk scores is a valuable approach in genetic epidemiology and increases the predictability of human quantitative traits based on genomic information compared with single SNP approach. PMID:20162742

  19. 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

    PubMed Central

    Kamerlin, Shina C. L.; Haranczyk, Maciej

    2009-01-01

    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 proper computational 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. Hydrolysis of oxaliplatin-evaluation of the acid dissociation constant for the oxalato monodentate complex.

    PubMed

    Jerremalm, Elin; Eksborg, Staffan; Ehrsson, Hans

    2003-02-01

    Alkaline hydrolysis of the platinum anticancer drug oxaliplatin gives the oxalato monodentate complex and the dihydrated oxaliplatin complex in two consecutive steps. The acid dissociation constant for the oxalato monodentate intermediate was determined by a kinetic approach. The pK(a) value was estimated as 7.23. The monodentate intermediate is assumed to rapidly react with endogenous compounds, resulting in a continuous conversion of oxaliplatin via the monodentate form. PMID:12532393

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. The testis-specific Cα2 subunit of PKA is kinetically indistinguishable from the common Cα1 subunit of PKA

    PubMed Central

    2011-01-01

    Background The two variants of the α-form of the catalytic (C) subunit of protein kinase A (PKA), designated Cα1 and Cα2, are encoded by the PRKACA gene. Whereas Cα1 is ubiquitous, Cα2 expression is restricted to the sperm cell. Cα1 and Cα2 are encoded with different N-terminal domains. In Cα1 but not Cα2 the N-terminal end introduces three sites for posttranslational modifications which include myristylation at Gly1, Asp-specific deamidation at Asn2 and autophosphorylation at Ser10. Previous reports have implicated specific biological features correlating with these modifications on Cα1. Since Cα2 is not modified in the same way as Cα1 we tested if they have distinct biochemical activities that may be reflected in different biological properties. Results We show that Cα2 interacts with the two major forms of the regulatory subunit (R) of PKA, RI and RII, to form cAMP-sensitive PKAI and PKAII holoenzymes both in vitro and in vivo as is also the case with Cα1. Moreover, using Surface Plasmon Resonance (SPR), we show that the interaction patterns of the physiological inhibitors RI, RII and PKI were comparable for Cα2 and Cα1. This is also the case for their potency to inhibit catalytic activities of Cα2 and Cα1. Conclusion We conclude that the regulatory complexes formed with either Cα1 or Cα2, respectively, are indistinguishable. PMID:21812984

  13. 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.

  14. 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.

  15. 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

  16. N4-monobutyryl-cCMP activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo.

    PubMed

    Wolter, Sabine; Dove, Stefan; Golombek, Marina; Schwede, Frank; Seifert, Roland

    2014-12-01

    There is increasing evidence for a role of cytidine 3',5'-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin(-) cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N(4)-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N(4)-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG. PMID:25192685

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

    PubMed

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

    2016-01-01

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

  18. 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

  19. Fat content, energy value and fatty acid profile of donkey milk during lactation and implications for human nutrition

    PubMed Central

    2012-01-01

    Background and aims Milk contains numerous nutrients. The content of n-3 fatty acids, the n-6/n-3 ratio, and short- and medium-chain fatty acids may promote positive health effects. In Western societies, cow’s milk fat is perceived as a risk factor for health because it is a source of a high fraction of saturated fatty acids. Recently, there has been increasing interest in donkey’s milk. In this work, the fat and energetic value and acidic composition of donkey’s milk, with reference to human nutrition, and their variations during lactation, were investigated. We also discuss the implications of the acidic profile of donkey’s milk on human nutrition. Methods Individual milk samples from lactating jennies were collected 15, 30, 45, 60, 90, 120, 150, 180 and 210days after foaling, for the analysis of fat, proteins and lactose, which was achieved using an infrared milk analyser, and fatty acids composition by gas chromatography. Results The donkey’s milk was characterised by low fat and energetic (1719.2kJ·kg-1) values, a high polyunsaturated fatty acids (PUFA) content of mainly α-linolenic acid (ALA) and linoleic acid (LA), a low n-6 to n-3 FA ratio or LA/ALA ratio, and advantageous values of atherogenic and thrombogenic indices. Among the minor PUFA, docosahesaenoic (DHA), eicosapentanoic (EPA), and arachidonic (AA) acids were present in very small amounts (<1%). In addition, the AA/EPA ratio was low (0.18). The fat and energetic values decreased (P < 0.01) during lactation. The fatty acid patterns were affected by the lactation stage and showed a decrease (P < 0.01) in saturated fatty acids content and an increase (P < 0.01) in the unsaturated fatty acids content. The n-6 to n-3 ratio and the LA/ALA ratio were approximately 2:1, with values <1 during the last period of lactation, suggesting the more optimal use of milk during this period. Conclusions The high level of unsaturated/saturated fatty acids and PUFA-n3 content and the low n-6/n-3 ratio

  20. Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products.

    PubMed

    Riedel, Sebastian L; Lu, Jingnan; Stahl, Ulf; Brigham, Christopher J

    2014-02-01

    Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here. PMID:24343766

  1. Using a Matlab Implemented Algorithm for UV-vis Spectral Resolution for pKa Determination and Multicomponent Analysis

    PubMed Central

    Gonen, Yotam; Rytwo, Giora

    2009-01-01

    A Matlab implemented computer code for spectral resolution is presented. The code enables the user to resolve the UV-visible absorption spectrum of a mixture of up to 3 previously known components, to the individual components, thus, evaluating their quantities. The resolving procedure is based on searching the combination of the components which yields the spectrum which is the most similar (minimal RMSE) to the measured spectrum of the mixture. Examples of using the software for pKa value estimation and multicomponent analysis are presented and other implementations are suggested. PMID:20072668

  2. Real-time relationship between PKA biochemical signal network dynamics and increased action potential firing rate in heart pacemaker cells: Kinetics of PKA activation in heart pacemaker cells.

    PubMed

    Yaniv, Yael; Ganesan, Ambhighainath; Yang, Dongmei; Ziman, Bruce D; Lyashkov, Alexey E; Levchenko, Andre; Zhang, Jin; Lakatta, Edward G

    2015-09-01

    cAMP-PKA protein kinase is a key nodal signaling pathway that regulates a wide range of heart pacemaker cell functions. These functions are predicted to be involved in regulation of spontaneous action potential (AP) generation of these cells. Here we investigate if the kinetics and stoichiometry of increase in PKA activity match the increase in AP firing rate in response to β-adrenergic receptor (β-AR) stimulation or phosphodiesterase (PDE) inhibition, that alters the AP firing rate of heart sinoatrial pacemaker cells. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, the EC50 in response to graded increases in the intensity of β-AR stimulation (by Isoproterenol) the magnitude of the increases in PKA activity and the spontaneous AP firing rate were similar (0.4±0.1nM vs. 0.6±0.15nM, respectively). Moreover, the kinetics (t1/2) of the increases in PKA activity and spontaneous AP firing rate in response to β-AR stimulation or PDE inhibition were tightly linked. We characterized the system rate-limiting biochemical reactions by integrating these experimentally derived data into a mechanistic-computational model. Model simulations predicted that phospholamban phosphorylation is a potent target of the increase in PKA activity that links to increase in spontaneous AP firing rate. In summary, the kinetics and stoichiometry of increases in PKA activity in response to a physiological (β-AR stimulation) or pharmacological (PDE inhibitor) stimuli match those of changes in the AP firing rate. Thus Ca(2+)-cAMP/PKA-dependent phosphorylation limits the rate and magnitude of increase in spontaneous AP firing rate. PMID:26241846

  3. 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…

  4. 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

  5. LKB1/AMPK and PKA Control ABCB11 Trafficking and Polarization in Hepatocytes

    PubMed Central

    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

  6. 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

  7. Sustained exposure to catecholamines affects cAMP/PKA compartmentalised signalling in adult rat ventricular myocytes.

    PubMed

    Fields, Laura A; Koschinski, Andreas; Zaccolo, Manuela

    2016-07-01

    In the heart compartmentalisation of cAMP/protein kinase A (PKA) signalling is necessary to achieve a specific functional outcome in response to different hormonal stimuli. Chronic exposure to catecholamines is known to be detrimental to the heart and disrupted compartmentalisation of cAMP signalling has been associated to heart disease. However, in most cases it remains unclear whether altered local cAMP signalling is an adaptive response, a consequence of the disease or whether it contributes to the pathogenetic process. We have previously demonstrated that isoforms of PKA expressed in cardiac myocytes, PKA-I and PKA-II, localise to different subcellular compartments and are selectively activated by spatially confined pools of cAMP, resulting in phosphorylation of distinct downstream targets. Here we investigate cAMP signalling in an in vitro model of hypertrophy in primary adult rat ventricular myocytes. By using a real time imaging approach and targeted reporters we find that that sustained exposure to catecholamines can directly affect cAMP/PKA compartmentalisation. This appears to involve a complex mechanism including both changes in the subcellular localisation of individual phosphodiesterase (PDE) isoforms as well as the relocalisation of PKA isoforms. As a result, the preferential coupling of PKA subsets with different PDEs is altered resulting in a significant difference in the level of cAMP the kinase is exposed to, with potential impact on phosphorylation of downstream targets. PMID:26475678

  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. PMID:27357676

  9. 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. PMID:26971161

  10. PKA Controls Calcium Influx into Motor Neurons during a Rhythmic Behavior

    PubMed Central

    Wang, Han; Sieburth, Derek

    2013-01-01

    Cyclic adenosine monophosphate (cAMP) has been implicated in the execution of diverse rhythmic behaviors, but how cAMP functions in neurons to generate behavioral outputs remains unclear. During the defecation motor program in C. elegans, a peptide released from the pacemaker (the intestine) rhythmically excites the GABAergic neurons that control enteric muscle contractions by activating a G protein-coupled receptor (GPCR) signaling pathway that is dependent on cAMP. Here, we show that the C. elegans PKA catalytic subunit, KIN-1, is the sole cAMP target in this pathway and that PKA is essential for enteric muscle contractions. Genetic analysis using cell-specific expression of dominant negative or constitutively active PKA transgenes reveals that knockdown of PKA activity in the GABAergic neurons blocks enteric muscle contractions, whereas constitutive PKA activation restores enteric muscle contractions to mutants defective in the peptidergic signaling pathway. Using real-time, in vivo calcium imaging, we find that PKA activity in the GABAergic neurons is essential for the generation of synaptic calcium transients that drive GABA release. In addition, constitutively active PKA increases the duration of calcium transients and causes ectopic calcium transients that can trigger out-of-phase enteric muscle contractions. Finally, we show that the voltage-gated calcium channels UNC-2 and EGL-19, but not CCA-1 function downstream of PKA to promote enteric muscle contractions and rhythmic calcium influx in the GABAergic neurons. Thus, our results suggest that PKA activates neurons during a rhythmic behavior by promoting presynaptic calcium influx through specific voltage-gated calcium channels. PMID:24086161

  11. 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.

  12. Differential modulation of large-conductance KCa channels by PKA in pregnant and nonpregnant myometrium.

    PubMed

    Pérez, G; Toro, L

    1994-05-01

    Uterine excitability depends on ion channel activity, the expression of which is regulated by sexual hormones. We show now that the action of protein kinase A (PKA) on large-conductance calcium-activated K+ (KCa) channel activity also depends on the hormonal status. PKA-dependent phosphorylation of reconstituted KCa channels from midpregnant rats usually stimulated channel activity; in contrast, KCa channels from nonpregnant rat and human myometrium were primarily inhibited by this mechanism. Both effects were reversible by phosphatase treatment. These results suggest that one important factor modulating uterine contractility during pregnancy or the regular cycle may be the differential response of KCa channels toward PKA-induced phosphorylation. PMID:7515569

  13. 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.

  14. [Clinical value of antibodies to lysobisphosphatidic acid in patients with primary antiphospholipid syndrome].

    PubMed

    Olivieri, S; Ruffatti, A; Bontadi, A; Cavazzana, A; Salvan, E; Cuffaro, S; Giunco, S; Punzi, L

    2010-01-01

    To assess the clinical value of anti-lysobisphosphatidic acid (anti-LBPA) antibodies in patients with primary antiphospholipid syndrome (APS), the sera of 140 primary APS patients were tested and compared with those of 70 control subjects affected with rheumatic systemic diseases (n. 24) or autoimmune thyroiditis (n. 46). Anti-LBPA anticardiolipin (aCL) and anti-beta2 Glycoprotein I (anti-beta2GPI) antibodies were determined using a "home made" ELISA method. Lupus anticoagulant (LA) was assessed using a series of clotting tests in accordance with the literature. IgG anti-LBPA was significantly prevalent in primary APS (p=0.000) with a sensitivity of 58.6% and a specificity of 92.9%. IgM anti-LBPA showed a significant frequency in primary APS (p=0.000) with a sensitivity of 28.6% and a specificity of 97.1%. Anti-LBPA's sensitivity and specificity for APS were lower or equal to those of aCL and anti-beta2GPI. The prevalence of anti-LBPA in the different clinical and laboratory subsets of APS was lower than those of aCL and anti-beta2GPI. It is interesting to observe that both IgG and IgM anti-LBPA were never found alone. The comparison between anti-LBPA and LA showed that the former had a higher sensitivity but a lower specificity. In conclusion, in view of our results anti-LBPA cannot at present be considered a further tool to be utilized to diagnose APS and to differentiate the different clinical and laboratory subsets of this disease. PMID:20657887

  15. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET.

    PubMed

    Lee, Shin-Rong; Sang, Lingjie; Yue, David T

    2016-03-29

    Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC), and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R) on PKA's catalytic subunit. We discover that this mutation not only differentially affects PKAcat's binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET. PMID:26997269

  16. 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

  17. 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. PMID:25782756

  18. Aripiprazole Increases the PKA Signalling and Expression of the GABAA Receptor and CREB1 in the Nucleus Accumbens of Rats.

    PubMed

    Pan, Bo; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

    2016-05-01

    The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling. PMID:26894264

  19. 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

  20. 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

  1. 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.

  2. Hedgehog induces formation of PKA-Smoothened complexes to promote Smoothened phosphorylation and pathway activation

    PubMed Central

    Li, Shuang; Ma, Guoqiang; Wang, Bing; Jiang, Jin

    2015-01-01

    Hedgehog (Hh) is a secreted glycoprotein that binds its receptor Patched to activate the G protein-coupled receptor-like protein Smoothened (Smo). In Drosophila, protein kinase A (PKA) phosphorylates and activates Smo in cells stimulated with Hh. In unstimulated cells, PKA phosphorylates and inhibits the transcription factor Cubitus interruptus (Ci). Here, we found that in cells exposed to Hh, the catalytic subunit of PKA (PKAc) bound to the juxtamembrane region of the C terminus of Smo. PKA-mediated phosphorylation of Smo further enhanced its association with PKAc to form stable kinase-substrate complexes that promoted the PKA-mediated trans-phosphorylation of Smo dimers. We identified multiple basic residues in the C-terminus of Smo that were required for interaction with PKAc, Smo phosphorylation, and Hh pathway activation. Hh induced a switch from the association of PKAc with a cytosolic complex of Ci and the kinesin-like protein Costal2 (Cos2) to a membrane-bound Smo-Cos2 complex. Thus, our study uncovers a previously uncharacterized mechanism for regulation of PKA activity and demonstrates that the signal-regulated formation of kinase-substrate complexes plays a central role in Hh signal transduction. PMID:24985345

  3. 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.

  4. Thermodynamic principles for the engineering of pH-driven conformational switches and acid insensitive proteins

    PubMed Central

    Bell-Upp, Peregrine; Robinson, Aaron C.; Whitten, Steven; Wheeler, Erika L.; Lin, Janine; Stites, Wesley E.; García-Moreno E, Bertrand

    2012-01-01

    The general thermodynamic principles behind pH driven conformational transitions of biological macromolecules are well understood. What is less obvious is how they can be used to engineer pH switches in proteins. The acid unfolding of staphylococcal nuclease (SNase) was used to illustrate different factors that can affect pH-driven conformational transitions. Acid unfolding is a structural transition driven by preferential H+ binding to the acid unfolded state (U) over the native (N) state of a protein. It is the result of carboxylic groups that titrate with more normal pKa values in the U state than in the N state. Acid unfolding profiles of proteins reflect a balance between electrostatic and non-electrostatic contributions to stability. Several strategies were used in attempts to turn SNase into an acid insensitive protein: (1) enhancing global stability of the protein with mutagenesis or with osmolytes, (2) use of high salt concentrations to screen Coulomb interactions, (3) stabilizing the N state through specific anion effects, (4) removing Asp or Glu residues that titrate with depressed pKa values in the N state, and (5) removing basic residues that might have strong repulsive interactions in the N state at low pH. The only effective way to engineer acid resistance in SNase is not through modulation of pKa values of Asp/Glu but by enhancing the global stability of the protein. Modulation of pH-driven conformational transitions by selective manipulation of the electrostatic component of the switch is an extremely difficult undertaking. PMID:21802194

  5. Pycnogenol supplementation promotes lipolysis via activation of cAMP-dependent PKA in ob/ob mice and primary-cultured adipocytes.

    PubMed

    Ho, Jin-Nyoung; Kim, Ok-Kyung; Nam, Da-Eun; Jun, Woojin; Lee, Jeongmin

    2014-01-01

    This study investigated the PKA-dependent inhibitory effect of pycnogenol (Pyc) on lipolysis using ob/ob mice and primary mouse adipocytes. Supplementation of Pyc at 30 mg/kg significantly reduced body weight gain and visceral fat mass. The serum and hepatic triglyceride (TG) and total cholesterol (TC) levels were reduced by Pyc supplementation, and high density lipoprotein (HDL)-cholesterol level significantly increased. In addition, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) mRNA levels increased with Pyc supplementation in adipose tissue of ob/ob mice. The treatment of primary cultured adipocytes with Pyc at 100 μg/mL significantly increased glycerol release, cAMP level by reduction of phosphodiestersae-3B (PDE3B), and HSL levels, but decreased protein levels of perilipin A and fatty acid synthetase (FAS). The PKA inhibitor (H89) clearly blocked the cellular levels of perilipin A and HSL, suggesting that Pyc promotes lipolysis of adipocytes through activation of cAMP-dependent PKA, resulting in induction of HSL and reduction of perilipin A. Therefore, this study may elucidate the possible mechanism of Pyc, which is a candidate for weight loss through stimulation of lipolysis. PMID:25866307

  6. Reduced ATGL-mediated lipolysis attenuates β-adrenergic-induced AMPK signaling, but not the induction of PKA-targeted genes, in adipocytes and adipose tissue.

    PubMed

    MacPherson, Rebecca E K; Dragos, Steven M; Ramos, Sofhia; Sutton, Charles; Frendo-Cumbo, Scott; Castellani, Laura; Watt, Matthew J; Perry, Christopher G R; Mutch, David M; Wright, David C

    2016-08-01

    5'-AMP-activated protein kinase (AMPK) is activated as a consequence of lipolysis and has been shown to play a role in regulation of adipose tissue mitochondrial content. Conversely, the inhibition of lipolysis has been reported to potentiate the induction of protein kinase A (PKA)-targeted genes involved in the regulation of oxidative metabolism. The purpose of the current study was to address these apparent discrepancies and to more fully examine the relationship between lipolysis, AMPK, and the β-adrenergic-mediated regulation of gene expression. In 3T3-L1 adipocytes, the adipose tissue triglyceride lipase (ATGL) inhibitor ATGListatin attenuated the Thr(172) phosphorylation of AMPK by a β3-adrenergic agonist (CL 316,243) independent of changes in PKA signaling. Similarly, CL 316,243-induced increases in the Thr(172) phosphorylation of AMPK were reduced in adipose tissue from whole body ATGL-deficient mice. Despite reductions in the activation of AMPK, the induction of PKA-targeted genes was intact or, in some cases, increased. Similarly, markers of mitochondrial content and respiration were increased in adipose tissue from ATGL knockout mice independent of changes in the Thr(172) phosphorylation of AMPK. Taken together, our data provide evidence that AMPK is not required for the regulation of adipose tissue oxidative capacity in conditions of reduced fatty acid release. PMID:27357546

  7. Synthesis, structure, and acid-base and redox properties of a family of new Ru(II) isomeric complexes containing the trpy and the dinucleating Hbpp ligands.

    PubMed

    Sens, Cristina; Rodríguez, Montserrat; Romero, Isabel; Llobet, Antoni; Parella, Teodor; Benet-Buchholz, Jordi

    2003-12-15

    Three pairs of mononuclear geometrical isomers containing the ligand 3,5-bis(2-pyridyl)pyrazole (Hbpp) of general formula in- and out-[RuII(Hbpp)(trpy)X](n+) (trpy=2,2':6',2' '-terpyridine; X=Cl, n=1, 2a,b; X=H2O, n=2, 3a,b; X=py (pyridine), n=2, 4a,b) have been prepared through two different synthetic routes, isolated, and structurally characterized. The solid state structural characterization was performed by X-ray diffraction analysis of four complexes: 2a-4a and 4b. The structural characterization in solution was performed by means of 1D and 2D NMR spectroscopy for complexes 2a,b and 4a,b and coincides with the structures found in the solid state. All complexes were also spectroscopically characterized by UV-vis which also allowed us to carry out spectrophotometric acid-base titrations. Thus, a number of species were spectroscopically characterized with the same oxidation state but with a different degree of protonation. As an example, for 3a three pKa values were obtained: pKa1(RuII)=2.13, pKa2(RuII)=6.88, and pKa3(RuII)=11.09. The redox properties were also studied, giving in all cases a number of electron transfers coupled to proton transfers. The pH dependency of the redox potentials allowed us to calculate the pKa of the complexes in the Ru(III) oxidation state. For complex 3a, these were found to be pKa1(RuIII)=0.01, pKa2(RuIII)=2.78, and pKa3(RuIII)=5.43. The oxidation state Ru(IV) was only reached from the Ru-OH2 type of complexes 3a or 3b. It has also been shown that the RuIV=O species derived from 3a is capable of electrocatalytically oxidizing benzyl alcohol with a second-order rate constant of kcat=17.1 M(-1) s(-1). PMID:14658892

  8. L-Stepholidine rescues memory deficit and synaptic plasticity in models of Alzheimer's disease via activating dopamine D1 receptor/PKA signaling pathway

    PubMed Central

    Hao, J-R; Sun, N; Lei, L; Li, X-Y; Yao, B; Sun, K; Hu, R; Zhang, X; Shi, X-D; Gao, C

    2015-01-01

    It is accepted that amyloid β-derived diffusible ligands (ADDLs) have a prominent role in triggering the early cognitive deficits that constitute Alzheimer's disease (AD). However, there is still no effective treatment for preventing or reversing the progression of the disease. Targeting α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor trafficking and its regulation is a new strategy for AD early treatment. Here we investigate the effect and mechanism of L-Stepholidine (L-SPD), which elicits dopamine D1-type receptor agonistic activity, while acting as D2-type receptor antagonist on cognition and synaptic plasticity in amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic (APP/PS1) mice, and hippocampal cultures or slices treated with ADDLs. L-SPD could improve the hippocampus-dependent memory, surface expression of glutamate receptor A (GluA1)-containing AMPA receptors and spine density in hippocampus of APP/PS1 transgenic mice. L-SPD not only rescued decreased phosphorylation and surface expression of GluA1 in hippocampal cultures but also protected the long-term potentiation in hippocampal slices induced by ADDLs. Protein kinase A (PKA) agonist Sp-cAMPS or D1-type receptor agonist SKF81297 had similar effects, whereas PKA antagonist Rp-cAMPS or D1-type receptor antagonist SCH23390 abolished the effect of L-SPD on GluA1 trafficking. This was mediated mainly by PKA, which could phosphorylate serine residue at 845 of the GluA1. L-SPD may be explored as a potential therapeutic drug for AD through a mechanism that improves AMPA receptor trafficking and synaptic plasticity via activating D1/PKA signaling pathway. PMID:26539912

  9. 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

  10. Substrate binding preferences and pKa determinations of a nitrile hydratase model complex: Variable solvent coordination to [(bmmp-TASN)Fe]OTf

    PubMed Central

    O’Toole, Martin G.; Bennett, Brian; Mashuta, Mark S.; Grapperhaus, Craig A.

    2009-01-01

    The five-coordinate iron-dithiolate complex (N,N′-4,7-bis-(2′-methyl-2′-mercatopropyl)-1-thia-4,7-diazacyclononane)iron(III), [LFe]+, has been isolated as the triflate salt from reaction of the previously reported LFeCl with thallium triflate. Spectroscopic characterization confirms an S = 1/2 ground state in non-coordinating solvents with room temperature µeff = 1.78 µB and EPR derived g-values of g1 = 2.06, g2 = 2.03 and g3 = 2.02. [LFe]+ binds a variety of coordinating solvents resulting in six-coordinate complexes [LFe-solvent]+. In acetonitrile the low-spin [LFe-NCMe]+ (g1 = 2.27, g2 = 2.18 and g3 = 1.98) is in equilibrium with [LFe]+ with a binding constant of Keq = 4.03 at room temperature. Binding of H2O, DMF, methanol, DMSO and pyridine to [LFe]+ yields high-spin six-coordinate complexes with EPR spectra that display significant strain in the rhombic zero-field splitting term E/D. Addition of one equivalent of triflic acid to the previously reported diiron species (LFe)2O results in the formation of [(LFe)2OH]OTf, which has been characterized by x-ray crystallography. The aqueous chemistry of [LFe]+ reveals three distinct species as a function of pH: [LFe-OH2]+, [(LFe)2OH]OTf, and (LFe)2O. The pKa values for [LFe-OH2]+ and [(LFe)2OH]OTf are 5.4 ± .1 and 6.52 ± .05 respectively. PMID:19166306

  11. 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

  12. 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

  13. 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...

  14. 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...

  15. 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...

  16. 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 ...

  17. 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. PMID:23510141

  18. 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

  19. 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.

  20. Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling.

    PubMed

    Krishnamurthy, Srinath; Moorthy, Balakrishnan Shenbaga; Xin Xiang, Lim; Xin Shan, Lim; Bharatham, Kavitha; Tulsian, Nikhil Kumar; Mihalek, Ivana; Anand, Ganesh S

    2014-09-16

    Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and

  1. PKA-type I selective constrained peptide disruptors of AKAP complexes.

    PubMed

    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

    2015-06-19

    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

  2. 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

  3. 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

  4. Identification of novel transcriptional regulators of PKA subunits in Saccharomyces cerevisiae by quantitative promoter-reporter screening.

    PubMed

    Pautasso, Constanza; Reca, Sol; Chatfield-Reed, Kate; Chua, Gordon; Galello, Fiorella; Portela, Paula; Zaremberg, Vanina; Rossi, Silvia

    2016-08-01

    The cAMP-dependent protein kinase (PKA) signaling is a broad pathway that plays important roles in the transduction of environmental signals triggering precise physiological responses. However, how PKA achieves the cAMP-signal transduction specificity is still in study. The regulation of expression of subunits of PKA should contribute to the signal specificity. Saccharomyces cerevisiae PKA holoenzyme contains two catalytic subunits encoded by TPK1, TPK2 and TPK3 genes, and two regulatory subunits encoded by BCY1 gene. We studied the activity of these gene promoters using a fluorescent reporter synthetic genetic array screen, with the goal of systematically identifying novel regulators of expression of PKA subunits. Gene ontology analysis of the identified modulators showed enrichment not only in the category of transcriptional regulators, but also in less expected categories such as lipid and phosphate metabolism. Inositol, choline and phosphate were identified as novel upstream signals that regulate transcription of PKA subunit genes. The results support the role of transcription regulation of PKA subunits in cAMP specificity signaling. Interestingly, known targets of PKA phosphorylation are associated with the identified pathways opening the possibility of a reciprocal regulation. PKA would be coordinating different metabolic pathways and these processes would in turn regulate expression of the kinase subunits. PMID:27188886

  5. Understanding the Highly Varying pKa of Arylamines. A Perspective from the Average Local Ionization Condensed-to-Atom Framework.

    PubMed

    Chamorro, Eduardo; Duque-Noreña, Mario

    2015-07-23

    The highly varying experimental pKa values for 36 arylamines spanning 7 orders of magnitude is carefully examined. Within this framework, a valence condensed-to-atom model for the average ionization energy is introduced and tested. The theoretical approach is connected to orbital Fukui functions directly mapped into semilocal or regional site-specific responses. It is revealed that the average local ionization energies associated with the amino nitrogen atom is linearly correlated to the basicity of the substituted arylamines, properly reproducing the experimental ordering of basicity. The condensed-to-atom descriptor exhibits a high predictive power, providing a new direct reactivity evaluation of significant value. PMID:26107310

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

    PubMed

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

    2015-01-01

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

  7. 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.)

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration

    PubMed Central

    Watson, Richard L.; Buck, Jochen; Levin, Lonny R.; Winger, Ryan C.; Wang, Jing; Arase, Hisashi

    2015-01-01

    CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM. PMID:26101266

  13. 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

  14. Impaired degradation of WNK by Akt and PKA phosphorylation of KLHL3.

    PubMed

    Yoshizaki, Yuki; Mori, Yutaro; Tsuzaki, Yoshihito; Mori, Takayasu; Nomura, Naohiro; Wakabayashi, Mai; Takahashi, Daiei; Zeniya, Moko; Kikuchi, Eriko; Araki, Yuya; Ando, Fumiaki; Isobe, Kiyoshi; Nishida, Hidenori; Ohta, Akihito; Susa, Koichiro; Inoue, Yuichi; Chiga, Motoko; Rai, Tatemitsu; Sasaki, Sei; Uchida, Shinichi; Sohara, Eisei

    2015-11-13

    Mutations in with-no-lysine kinase (WNK) 1, WNK4, Kelch-like 3 (KLHL3), and Cullin3 result in an inherited hypertensive disease, pseudohypoaldosteronism type II. WNK activates the Na-Cl cotransporter (NCC), increasing sodium reabsorption in the kidney. Further, KLHL3, an adapter protein of Cullin3-based E3 ubiquitin ligase, has been recently found to bind to WNK, thereby degrading them. Insulin and vasopressin have been identified as powerful activators of WNK signaling. In this study, we investigated effects of Akt and PKA, key downstream substrates of insulin and vasopressin signaling, respectively, on KLHL3. Mass spectrometry analysis revealed that KLHL3 phosphorylation at S433. Phospho-specific antibody demonstrated defective binding between phosphorylated KLHL3 and WNK4. Consistent with the fact that S433 is a component of Akt and PKA phosphorylation motifs, in vitro kinase assay demonstrated that Akt and PKA can phosphorylate KLHL3 at S433, that was previously reported to be phosphorylated by PKC. Further, forskolin, a representative PKA stimulator, increased phosphorylation of KLHL3 at S433 and WNK4 protein expression in HEK293 cells by inhibiting the KLHL3 effect that leads to WNK4 degradation. Insulin also increased phosphorylation of KLHL3 at S433 in cultured cells. In conclusion, we found that Akt and PKA phosphorylated KLHL3 at S433, and phosphorylation of KLHL3 by PKA inhibited WNK4 degradation. This could be a novel mechanism on how insulin and vasopressin physiologically activate the WNK signal. PMID:26435498

  15. The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

    PubMed Central

    Liang, J; Steinberg, G; Livnah, N; Sheves, M; Ebrey, T G; Tsuda, M

    1994-01-01

    A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled. PMID:7948697

  16. PKA Compartmentalization via AKAP220 and AKAP12 Contributes to Endothelial Barrier Regulation

    PubMed Central

    Radeva, Mariya Y.; Kugelmann, Daniela; Spindler, Volker; Waschke, Jens

    2014-01-01

    cAMP-mediated PKA signaling is the main known pathway involved in maintenance of the endothelial barrier. Tight regulation of PKA function can be achieved by discrete compartmentalization of the enzyme via physical interaction with A-kinase anchoring proteins (AKAPs). Here, we investigated the role of AKAPs 220 and 12 in endothelial barrier regulation. Analysis of human and mouse microvascular endothelial cells as well as isolated rat mesenteric microvessels was performed using TAT-Ahx-AKAPis peptide, designed to competitively inhibit PKA-AKAP interaction. In vivo microvessel hydraulic conductivity and in vitro transendothelial electrical resistance measurements showed that this peptide destabilized endothelial barrier properties, and dampened the cAMP-mediated endothelial barrier stabilization induced by forskolin and rolipram. Immunofluorescence analysis revealed that TAT-Ahx-AKAPis led to both adherens junctions and actin cytoskeleton reorganization. Those effects were paralleled by redistribution of PKA and Rac1 from endothelial junctions and by Rac1 inactivation. Similarly, membrane localization of AKAP220 was also reduced. In addition, depletion of either AKAP12 or AKAP220 significantly impaired endothelial barrier function and AKAP12 was also shown to interfere with cAMP-mediated barrier enhancement. Furthermore, immunoprecipitation analysis demonstrated that AKAP220 interacts not only with PKA but also with VE-cadherin and ß-catenin. Taken together, these results indicate that AKAP-mediated PKA subcellular compartmentalization is involved in endothelial barrier regulation. More specifically, AKAP220 and AKAP12 contribute to endothelial barrier function and AKAP12 is required for cAMP-mediated barrier stabilization. PMID:25188285

  17. Constitutively active PKA regulates neuronal acetylcholine release and contractility of guinea pig urinary bladder smooth muscle.

    PubMed

    Xin, Wenkuan; Li, Ning; Fernandes, Vitor S; Petkov, Georgi V

    2016-06-01

    Autonomic and somatic motor neurons that innervate the urinary bladder and urethra control the highly coordinated functions of the lower urinary tract, the storage, and the emptying of urine. ACh is the primary excitatory neurotransmitter in the bladder. Here, we aimed to determine whether PKA regulates neuronal ACh release and related nerve-evoked detrusor smooth muscle (DSM) contractions in the guinea pig urinary bladder. Isometric DSM tension recordings were used to measure spontaneous phasic and electrical field stimulation (EFS)- and carbachol-induced DSM contractions with a combination of pharmacological tools. The colorimetric method was used to measure ACh released by the parasympathetic nerves in DSM isolated strips. The pharmacological inhibition of PKA with H-89 (10 μM) increased the spontaneous phasic contractions, whereas it attenuated the EFS-induced DSM contractions. Intriguingly, H-89 (10 μM) attenuated the (primary) cholinergic component, whereas it simultaneously increased the (secondary) purinergic component of the nerve-evoked contractions in DSM isolated strips. The acetylcholinesterase inhibitor, eserine (10 μM), increased EFS-induced DSM contractions, and the subsequent addition of H-89 attenuated the contractions. H-89 (10 μM) significantly increased DSM phasic contractions induced by the cholinergic agonist carbachol. The inhibition of PKA decreased the neuronal release of ACh in DSM tissues. This study revealed that PKA-mediated signaling pathways differentially regulate nerve-evoked and spontaneous phasic contractions of guinea pig DSM. Constitutively active PKA in the bladder nerves controls synaptic ACh release, thus regulating the nerve-evoked DSM contractions, whereas PKA in DSM cells controls the spontaneous phasic contractility. PMID:27029424

  18. 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. PMID:21889401

  19. 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. PMID:25521813

  20. 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

  1. 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

  2. Preferential Phosphorylation of R-domain Serine 768 Dampens Activation of CFTR Channels by PKA

    PubMed Central

    Csanády, László; Seto-Young, Donna; Chan, Kim W.; Cenciarelli, Cristina; Angel, Benjamin B.; Qin, Jun; McLachlin, Derek T.; Krutchinsky, Andrew N.; Chait, Brian T.; Nairn, Angus C.; Gadsby, David C.

    2005-01-01

    CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The

  3. Normal values for nuclear cardiology: Japanese databases for myocardial perfusion, fatty acid and sympathetic imaging and left ventricular function.

    PubMed

    Nakajima, Kenichi

    2010-04-01

    Myocardial normal databases for stress myocardial perfusion study have been created by the Japanese Society of Nuclear Medicine Working Group. The databases comprised gender-, camera rotation range- and radiopharmaceutical-specific data-sets from multiple institutions, and normal database files were created for installation in common nuclear cardiology software. Based on the electrocardiography-gated single-photon emission computed tomography (SPECT), left ventricular function, including ventricular volumes, systolic and diastolic functions and systolic wall thickening were also analyzed. Normal databases for fatty acid imaging using (123)I-beta-methyl-iodophenyl-pentadecanoic acid and sympathetic imaging using (123)I-meta-iodobenzylguanidine were also examined. This review provides lists and overviews of normal values for myocardial SPECT and ventricular function in a Japanese population. The population-specific approach is a key factor for proper diagnostic and prognostic evaluation. PMID:20108130

  4. 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

  5. Normal values for nuclear cardiology: Japanese databases for myocardial perfusion, fatty acid and sympathetic imaging and left ventricular function

    PubMed Central

    2010-01-01

    Myocardial normal databases for stress myocardial perfusion study have been created by the Japanese Society of Nuclear Medicine Working Group. The databases comprised gender-, camera rotation range- and radiopharmaceutical-specific data-sets from multiple institutions, and normal database files were created for installation in common nuclear cardiology software. Based on the electrocardiography-gated single-photon emission computed tomography (SPECT), left ventricular function, including ventricular volumes, systolic and diastolic functions and systolic wall thickening were also analyzed. Normal databases for fatty acid imaging using 123I-beta-methyl-iodophenyl-pentadecanoic acid and sympathetic imaging using 123I-meta-iodobenzylguanidine were also examined. This review provides lists and overviews of normal values for myocardial SPECT and ventricular function in a Japanese population. The population-specific approach is a key factor for proper diagnostic and prognostic evaluation. PMID:20108130

  6. Suitability of hyperspectral imaging for rapid evaluation of thiobarbituric acid (TBA) value in grass carp (Ctenopharyngodon idella) fillet.

    PubMed

    Cheng, Jun-Hu; Sun, Da-Wen; Pu, Hong-Bin; Wang, Qi-Jun; Chen, Yu-Nan

    2015-03-15

    The suitability of hyperspectral imaging technique (400-1000 nm) was investigated to determine the thiobarbituric acid (TBA) value for monitoring lipid oxidation in fish fillets during cold storage at 4°C for 0, 2, 5, and 8 days. The PLSR calibration model was established with full spectral region between the spectral data extracted from the hyperspectral images and the reference TBA values and showed good performance for predicting TBA value with determination coefficients (R(2)P) of 0.8325 and root-mean-square errors of prediction (RMSEP) of 0.1172 mg MDA/kg flesh. Two simplified PLSR and MLR models were built and compared using the selected ten most important wavelengths. The optimised MLR model yielded satisfactory results with R(2)P of 0.8395 and RMSEP of 0.1147 mg MDA/kg flesh, which was used to visualise the TBA values distribution in fish fillets. The whole results confirmed that using hyperspectral imaging technique as a rapid and non-destructive tool is suitable for the determination of TBA values for monitoring lipid oxidation and evaluation of fish freshness. PMID:25308667

  7. 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

  8. 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

  9. Accurate proton affinity and gas-phase basicity values for molecules important in biocatalysis

    PubMed Central

    Moser, Adam; Range, Kevin; York, Darrin M.

    2010-01-01

    Benchmark quantum calculations of proton affinities and gas phase basicities of molecules relevant to biochemical processes, particulsarly acid/base catalysis, are presented and compared for a variety of multi-level and density-functional quantum models. Included are nucleic acid bases in both keto and enol tautomeric forms, ribose in B-form and A-form sugar pucker conformations, amino acid side chains and backbone molecules, and various phosphates and phosphoranes including thio substitutions. This work presents a high-level thermodynamic characterization of biologically relevant protonation states, and provides a benchmark database for development of next-generation semiempirical and approximate density-functional quantum models, and parameterization of methods to predict pKa values and relative solvation energies. PMID:20942500

  10. Valorization of biodiesel derived glycerol as a carbon source to obtain added-value metabolites: Focus on polyunsaturated fatty acids.

    PubMed

    Abad, Sergi; Turon, Xavier

    2012-01-01

    The amount of glycerol derived from the biodiesel industry is exponentially increasing. The valorization of glycerol has acquired attention and resources with an obvious economic and environmental interest. Glycerol has the potential to improve the profitability of biodiesel in a biorefinery scenario. Added-value metabolites obtained from glycerol-based fermentations are the target of multiple research studies, primarily chemicals and biopolymers. Pigments and polyunsaturated fatty acids are exceptional examples as they have market presence as nutraceuticals. Most of the studies reviewed have been based on microalgae cultures. Depending on the strain and the engineering aspects of such cultures the final yield suffers notable variations. This is an emerging field which shows great potential from the perspective of a byproduct usage and the increasing yields (value) obtained from the bioprocess. PMID:22261015

  11. 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. PMID:25086614

  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. PMID:26117239

  13. 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.

  14. AKAP-Anchored PKA Maintains Neuronal L-type Calcium Channel Activity and NFAT Transcriptional Signaling

    PubMed Central

    Murphy, Jonathan G.; Sanderson, Jennifer L.; Gorski, Jessica A.; Scott, John D.; Catterall, William A.; Sather, William A.; Dell’Acqua, Mark L.

    2014-01-01

    Summary In neurons, Ca2+ influx through L-type voltage-gated Ca2+ channels (LTCC) couples electrical activity to changes in transcription. LTCC activity is elevated by the cAMP-dependent protein kinase (PKA) and depressed by the Ca2+-dependent phosphatase calcineurin (CaN), with both enzymes localized to the channel by A-kinase anchoring protein (AKAP) 79/150. AKAP79/150 anchoring of CaN also promotes LTCC activation of transcription through dephosphorylation of the nuclear factor of activated T-cells (NFAT). We report here that genetic disruption of PKA anchoring to AKAP79/150 also interferes with LTCC activation of CaN-NFAT signaling in neurons. Disruption of AKAP-PKA anchoring promoted redistribution of the kinase out of dendritic spines, profound decreases in LTCC phosphorylation and Ca2+ influx, and impaired NFAT movement to the nucleus and activation of transcription. Our findings support a model wherein basal activity of AKAP79/150-anchored PKA opposes CaN to preserve LTCC phosphorylation, thereby sustaining LTCC activation of CaN-NFAT signaling to the neuronal nucleus. PMID:24835999

  15. 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. PMID:17266791

  16. 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

  17. 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. PMID:25319570

  18. 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. PMID:25376656

  19. Activation of the cAMP-PKA pathway Antagonizes Metformin Suppression of Hepatic Glucose Production.

    PubMed

    He, Ling; Chang, Evan; Peng, Jinghua; An, Hongying; McMillin, Sara M; Radovick, Sally; Stratakis, Constantine A; Wondisford, Fredric E

    2016-05-13

    Metformin is the most commonly prescribed oral anti-diabetic agent worldwide. Surprisingly, about 35% of diabetic patients either lack or have a delayed response to metformin treatment, and many patients become less responsive to metformin over time. It remains unknown how metformin resistance or insensitivity occurs. Recently, we found that therapeutic metformin concentrations suppressed glucose production in primary hepatocytes through AMPK; activation of the cAMP-PKA pathway negatively regulates AMPK activity by phosphorylating AMPKα subunit at Ser-485, which in turn reduces AMPK activity. In this study, we find that metformin failed to suppress glucose production in primary hepatocytes with constitutively activated PKA and did not improve hyperglycemia in mice with hyperglucagonemia. Expression of the AMPKα1(S485A) mutant, which is unable to be phosphorylated by PKA, increased both AMPKα activation and the suppression of glucose production in primary hepatocytes treated with metformin. Intriguingly, salicylate/aspirin prevents the phosphorylation of AMPKα at Ser-485, blocks cAMP-PKA negative regulation of AMPK, and improves metformin resistance. We propose that aspirin/salicylate may augment metformin's hepatic action to suppress glucose production. PMID:27002150

  20. Differential effects of PKA-controlled CaMKK2 variants on neuronal differentiation

    PubMed Central

    Cao, Wenguang; Sohail, Muhammad; Liu, Guodong; Koumbadinga, Geremy A; Lobo, Vincent G

    2011-01-01

    Regulation between protein kinases is critical for the establishment of signaling pathways/networks to orchestrate cellular processes. Besides posttranslational phosphorylation, alternative pre-mRNA splicing is another way to control kinase properties, but splicing regulation between two kinases and the effect of resulting variants on cells have not been explored. We examined the effect of the protein kinase A (PKA) pathway on the alternative splicing and variant properties of the Ca++/calmodulin-dependent protein kinase kinase 2 (CaMKK2) gene in B35 neuroblastoma cells. Inclusion of the exon 16 of CaMKK2 was significantly reduced by H89, a PKA selective inhibitor. Consistently, overexpressed PKA strongly promoted the exon inclusion in a CaMKK2 sequence-dependent way in splicing reporter assays. In vitro, purified CaMKK2 variant proteins were kinase-active. In cells, they were differentially phosphorylated by PKA. In RNA interference assays, CaMKK2 was required for forskolin-induced neurite growth. Interestingly, overexpression of the variant without exon 16 (−E16) promoted neurite elongation while the other one (+E16) promoted neurite branching; in contrast, reduction of the latter variant enhanced neurite elongation. Moreover, the variants are differentially expressed and the exon 16-containing transcripts highly enriched in the brain, particularly the cerebellum and hippocampus. Thus, PKA regulates the alternative splicing of CaMKK2 to produce variants that differentially modulate neuronal differentiation. Taken together with the many distinct variants of kinases, alternative splicing regulation likely adds another layer of modulation between protein kinases in cellular signaling networks. PMID:21957496

  1. 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. PMID:26403272

  2. 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

  3. 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

  4. 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

  5. Molecule specific effects of PKA-mediated phosphorylation on rat isolated heart and cardiac myofibrillar function.

    PubMed

    Hanft, Laurin M; Cornell, Timothy D; McDonald, Colin A; Rovetto, Michael J; Emter, Craig A; McDonald, Kerry S

    2016-07-01

    Increased cardiac myocyte contractility by the β-adrenergic system is an important mechanism to elevate cardiac output to meet hemodynamic demands and this process is depressed in failing hearts. While increased contractility involves augmented myoplasmic calcium transients, the myofilaments also adapt to boost the transduction of the calcium signal. Accordingly, ventricular contractility was found to be tightly correlated with PKA-mediated phosphorylation of two myofibrillar proteins, cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI), implicating these two proteins as important transducers of hemodynamics to the cardiac sarcomere. Consistent with this, we have previously found that phosphorylation of myofilament proteins by PKA (a downstream signaling molecule of the beta-adrenergic system) increased force, slowed force development rates, sped loaded shortening, and increased power output in rat skinned cardiac myocyte preparations. Here, we sought to define molecule-specific mechanisms by which PKA-mediated phosphorylation regulates these contractile properties. Regarding cTnI, the incorporation of thin filaments with unphosphorylated cTnI decreased isometric force production and these changes were reversed by PKA-mediated phosphorylation in skinned cardiac myocytes. Further, incorporation of unphosphorylated cTnI sped rates of force development, which suggests less cooperative thin filament activation and reduced recruitment of non-cycling cross-bridges into the pool of cycling cross-bridges, a process that would tend to depress both myocyte force and power. Regarding MyBP-C, PKA treatment of slow-twitch skeletal muscle fibers caused phosphorylation of MyBP-C (but not slow skeletal TnI (ssTnI)) and yielded faster loaded shortening velocity and ∼30% increase in power output. These results add novel insight into the molecular specificity by which the β-adrenergic system regulates myofibrillar contractility and how attenuation of PKA

  6. 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

  7. Added value of hepatobiliary phase gadoxetic acid-enhanced MRI for diagnosing hepatocellular carcinoma in high-risk patients

    PubMed Central

    Phongkitkarun, Sith; Limsamutpetch, Kuruwin; Tannaphai, Penampai; Jatchavala, Janjira

    2013-01-01

    AIM: To determine the added value of hepatobiliary phase (HBP) gadoxetic acid-enhanced magnetic resonance imaging (MRI) in evaluating hepatic nodules in high-risk patients. METHODS: The institutional review board approved this retrospective study and waived the requirement for informed consent. This study included 100 patients at high risk for hepatocellular carcinoma (HCC) and 105 hepatic nodules that were larger than 1 cm. A blind review of two MR image sets was performed in a random order: set 1, unenhanced (T1- and T2-weighted) and dynamic images; and set 2, unenhanced, dynamic 20-min and HBP images. The diagnostic accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were compared for the two image sets. Univariate and multivariate analyses were performed on the MR characteristics utilized to diagnose HCC. RESULTS: A total of 105 hepatic nodules were identified in 100 patients. Fifty-nine nodules were confirmed to be HCC. The diameter of the 59 HCCs ranged from 1 to 12 cm (mean: 1.9 cm). The remaining 46 nodules were benign (28 were of hepatocyte origin, nine were hepatic cysts, seven were hemangiomas, one was chronic inflammation, and one was focal fat infiltration). The diagnostic accuracy significantly increased with the addition of HBP images, from 88.7% in set 1 to 95.5% in set 2 (P = 0.002). In set 1 vs set 2, the sensitivity and NPV increased from 79.7% to 93.2% and from 78.9% to 91.8%, respectively, whereas the specificity and PPV were not significantly different. The hypointensity on the HBP images was the most sensitive (93.2%), and typical arterial enhancement followed by washout was the most specific (97.8%). The multivariate analysis revealed that typical arterial enhancement followed by washout, hyperintensity on T2-weighted images, and hypointensity on HBP images were statistically significant MRI findings that could diagnose HCC (P < 0.05). CONCLUSION: The addition of HBP gadoxetic acid

  8. 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. PMID:26256345

  9. Solvent effect on anthranilic acid spectroscopy.

    PubMed

    Abou-Zied, Osama K; Al-Busaidi, Badriya Y; Husband, John

    2014-01-01

    The spectroscopy of anthranilic acid (AA) was examined in neat and binary solvents of varying polarity and hydrogen bonding strength in order to understand the role of water in solvating the polar sites of the molecule. With the exception of water, the Stokes shift of AA in different solvents was found to be linearly correlated with the normalized molar transition energy of solvent polarity (ETN), indicating the major role of the hydrogen bonding effect in solution. Analysis of the absorption and fluorescence spectra reveals that AA exists as an anion in neutral water. The pKa (4.50) and pKa* (4.44) values were estimated from the spectral shift in the absorption and fluorescence spectra measured in different pH solutions. The shortest fluorescence lifetime was measured in cyclohexane and is attributed to intramolecular hydrogen dislocation/transfer in the excited state. The lifetime values in polar solvents point to the dominant effect of the hydrogen-bond donating strength (α value) of the solvent. The number of water molecules solvating the polar region of the neutral form of AA was estimated to be three from the absorbance change in dioxane/buffer (pH 3.5) binary mixtures. The structures of AA:water complexes were calculated from density functional theory using the B3LYP method with a 6-311++G(2d,p) basis set. A stepwise addition of water molecules (1–3) to the polar region of AA leads to a preferential solvation of the COOH group of the molecule in a closed-cyclic geometry. It is worth noting that the spectral shift as a function of pH suggests the suitability of AA as a probe to estimate the local acidity of binding sites in macromolecules in the pH range 3.0–7.0. PMID:24102373

  10. 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

  11. PACAP decides neuronal laminar fate via PKA signaling in the developing cerebral cortex

    SciTech Connect

    Ohtsuka, Masanari; Fukumitsu, Hidefumi Furukawa, Shoei

    2008-05-16

    Laminar formation in the developing cerebral cortex requires the precisely regulated generation of phenotype-specified neurons. To test the possible involvement of pituitary adenylate cyclase-activating polypeptide (PACAP) in this formation, we investigated the effects of PACAP administered into the telencephalic ventricular space of 13.5-day-old mouse embryos. PACAP partially inhibited the proliferation of cortical progenitors and altered the position and gene-expression profiles of newly generated neurons otherwise expected for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex. The former and latter effects were seen only when the parent progenitor cells were exposed to PACAP in the later and in earlier G1 phase, respectively; and these effects were suppressed by co-treatment with a protein kinase A (PKA) inhibitor. These observations suggest that PACAP participates in the processes forming the neuronal laminas in the developing cortex via the intracellular PKA pathway.

  12. In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs

    PubMed Central

    Röck, Ruth; Bachmann, Verena; Bhang, Hyo-eun C; Malleshaiah, Mohan; Raffeiner, Philipp; Mayrhofer, Johanna E; Tschaikner, Philipp M; Bister, Klaus; Aanstad, Pia; Pomper, Martin G; Michnick, Stephen W; Stefan, Eduard

    2015-01-01

    Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems. PMID:26099953

  13. 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

  14. Prostaglandin E2 Reverses Aberrant Production of an Inflammatory Chemokine by Microglia from Sandhoff Disease Model Mice through the cAMP-PKA Pathway

    PubMed Central

    Kawashita, Eri; Tsuji, Daisuke; Toyoshima, Masahiro; Kanno, Yosuke; Matsuno, Hiroyuki; Itoh, Kohji

    2011-01-01

    Background Sandhoff disease (SD) is a neurodegenerative lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including terminal GlcNAc-oligosaccharides and GM2 ganglioside. Microglia-mediated neuroinflammation contributes to the pathogenesis and progression of SD. Our previous study demonstrated that MIP-1α, a putative pathogenic factor for SD, is up-regulated in microglial cells derived from SD model mice (SD-Mg) through activation of Akt and JNK. Methodology/Principal Findings In this study, we first demonstrated that prostaglandin E2 (PGE2), which is one of the lipid mediators derived from arachidonic acid and is known to suppress activation of microglia, reduced the aberrant MIP-1α production by SD-Mg to the same level as by WT-Mg. PGE2 also attenuated the activation of Akt and JNK. The inhibition of MIP-1α production and the activation of Akt and JNK occurred through the EP2 and 4/cAMP/PKA signaling pathway in the murine microglia derived from SD model mice. Conclusions/Significance We propose that PGE2 plays a role as a negative regulator of MIP-1α production in the pathogenesis of SD, and that PGE2-EP2 and 4/cAMP/PKA signaling could be a target pathway for therapy for SD. PMID:21298000

  15. 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

  16. Regulation of proximal tubule vacuolar H+-ATPase by PKA and AMP-activated protein kinase

    PubMed Central

    Al-bataineh, Mohammad M.; Gong, Fan; Marciszyn, Allison L.; Myerburg, Michael M.

    2014-01-01

    The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress. PMID:24553431

  17. Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes☆

    PubMed Central

    Bryant, Simon; Kimura, Tomomi E.; Kong, Cherrie H.T.; Watson, Judy J.; Chase, Anabelle; Suleiman, M. Saadeh; James, Andrew F.; Orchard, Clive H.

    2014-01-01

    L-type Ca channels (LTCC), which play a key role in cardiac excitation–contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation. PMID:24412535

  18. Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes.

    PubMed

    Bryant, Simon; Kimura, Tomomi E; Kong, Cherrie H T; Watson, Judy J; Chase, Anabelle; Suleiman, M Saadeh; James, Andrew F; Orchard, Clive H

    2014-03-01

    L-type Ca channels (LTCC), which play a key role in cardiac excitation-contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation. PMID:24412535

  19. 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.

  20. 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

  1. PKA-CREB-BDNF signaling regulated long lasting antidepressant activities of Yueju but not ketamine.

    PubMed

    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. Nutrient Control of Yeast Gametogenesis Is Mediated by TORC1, PKA and Energy Availability.

    PubMed

    Weidberg, Hilla; Moretto, Fabien; Spedale, Gianpiero; Amon, Angelika; van Werven, Folkert J

    2016-06-01

    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

  4. Differentially regulated protein kinase A (PKA) activity in adipose tissue and liver is associated with resistance to diet-induced obesity and glucose intolerance in mice that lack PKA regulatory subunit type IIα.

    PubMed

    London, Edra; Nesterova, Maria; Sinaii, Ninet; Szarek, Eva; Chanturiya, Tatyana; Mastroyannis, Spyridon A; Gavrilova, Oksana; Stratakis, Constantine A

    2014-09-01

    The cAMP-dependent protein kinase A (PKA) signaling system is widely expressed and has a central role in regulating cellular metabolism in all organ systems affected by obesity. PKA has four regulatory (RIα, RIIα, RIβ, RIIβ) and four catalytic (Cα, Cβ, Cγ, Prkx) subunit isoforms that have tissue-specific expression profiles. In mice, knockout (KO) of RIIβ, the primary PKA regulatory subunit in adipose tissue or knockout of the catalytic subunit Cβ resulted in a lean phenotype that resists diet-induced obesity and associated metabolic complications. Here we report that the disruption of the ubiquitously expressed PKA RIIα subunit in mice (RIIαKO) confers resistance to diet-induced obesity, glucose intolerance, and hepatic steatosis. After 2-week high-fat diet exposure, RIIαKO mice weighed less than wild-type littermates. Over time this effect was more pronounced in female mice that were also leaner than their wild-type counterparts, regardless of the diet. Decreased intake of a high-fat diet contributed to the attenuated weight gain in RIIαKO mice. Additionally, RIIα deficiency caused differential regulation of PKA in key metabolic organs: cAMP-stimulated PKA activity was decreased in liver and increased in gonadal adipose tissue. We conclude that RIIα represents a potential target for therapeutic interventions in obesity, glucose intolerance, and nonalcoholic fatty liver disease. PMID:24914943

  5. 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

  6. 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.

  7. 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

  8. Ca2+-regulated-cAMP/PKA signaling in cardiac pacemaker cells links ATP supply to demand

    PubMed Central

    Yaniv, Yael; Juhaszova, Magdalena; Lyashkov, Alexey E.; Spurgeon, Harold A.; Sollott, Steven J.; Lakatta, Edward G.

    2011-01-01

    Rationale In sinoatrial node cells (SANC), Ca2+ activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation of Ca2+ cycling proteins. These result in spontaneous sarcoplasmic-reticulum (SR) generated rhythmic Ca2+ oscillations during diastolic depolarization, that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA signaling. ATP is consumed to pump Ca2+ to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Objective Since a negative feedback mechanism links ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca2+-cAMP/PKA dependent; and (2) due to its feed–forward nature, a decrease in flux through the Ca2+-cAMP/PKA signaling axis will reduce the basal ATP production rate. Methods and Results O2 consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca2+-cAMP/PKA signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion (r2=0.96), and reduced O2 consumption and flavoprotein fluorescence. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca2+ flux reduced the ATP level. Conclusions Feed-forward basal Ca2+-cAMP/PKA signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca2+-cAMP/PKA signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this feed-forward basal cAMP/PKA signaling, and in which ATP level remains constant when the demand changes. PMID:21835182

  9. Parathyroid hormone induces adipocyte lipolysis via PKA-mediated phosphorylation of hormone-sensitive lipase.

    PubMed

    Larsson, Sara; Jones, Helena A; Göransson, Olga; Degerman, Eva; Holm, Cecilia

    2016-03-01

    Parathyroid hormone (PTH) is secreted from the parathyroid glands in response to low plasma calcium levels. Besides its classical actions on bone and kidney, PTH may have other important effects, including metabolic effects, as suggested for instance by increased prevalence of insulin resistance and type 2 diabetes in patients with primary hyperparathyroidism. Moreover, secondary hyperparathyroidism may contribute to the metabolic derangements that characterize states of vitamin D deficiency. PTH has been shown to induce adipose tissue lipolysis, but the details of the lipolytic action of PTH have not been described. Here we used primary mouse adipocytes to show that intact PTH (1-84) as well as the N-terminal fragment (1-37) acutely stimulated lipolysis in a dose-dependent manner, whereas the C-terminal fragment (38-84) was without lipolytic effect. The lipolytic action of PTH was paralleled by phosphorylation of known protein kinase A (PKA) substrates, i.e. hormone-sensitive lipase (HSL) and perilipin. The phosphorylation of HSL in response to PTH occurred at the known PKA sites S563 and S660, but not at the non-PKA site S565. PTH-induced lipolysis, as well as phosphorylation of HSL at S563 and S660, was blocked by both the PKA-inhibitor H89 and the adenylate cyclase inhibitor MDL-12330A, whereas inhibitors of extracellular-regulated kinase (ERK), protein kinase B (PKB), AMP-activated protein kinase (AMPK) and Ca(2+)/calmodulin-dependent protein kinase (CaMK) had little or no effect. Inhibition of phosphodiesterase 4 (PDE4) strongly potentiated the lipolytic action of PTH, whereas inhibition of PDE3 had no effect. Our results show that the lipolytic action of PTH is mediated by the PKA signaling pathway with no or minor contribution of other signaling pathways and, furthermore, that the lipolytic action of PTH is limited by simultaneous activation of PDE4. Knowledge of the signaling pathways involved in the lipolytic action of PTH is important for our

  10. Behavioral expression of cocaine sensitization in rats is accompanied by a distinct pattern of modifications in the PKA/DARPP-32 signaling pathway.

    PubMed

    Scheggi, Simona; Raone, Anna; De Montis, Maria Graziella; Tagliamonte, Alessandro; Gambarana, Carla

    2007-11-01

    Repeated cocaine administration induces behavioral sensitization and modifications in the phosphorylation pattern of dopamine and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32), characterized by a tonic increase in the Thr75 phosphorylated form, and a decrease in the Thr34 phosphorylated form. This study further investigated the correlations between cocaine sensitization and modifications in the DARPP-32 phosphorylation pattern, cAMP-dependent protein kinase (PKA) activity, and mGluR5 tone in the medial prefrontal cortex and nucleus accumbens. Behavioral sensitization and modifications in these neurochemical markers followed a similar temporal pattern. Moreover, in sensitized rats acute cocaine administration modified phosphorylation levels of Thr75- and Thr34-DARPP-32, GluR1, and NR1 subunits in the nucleus accumbens only at a dose double the efficacious dose in control rats. These results suggest that the high levels of phospho-Thr75 DARPP-32 maintain PKA in a prevalent inhibited state. Furthermore, in sensitized rats the acute administration of 6-methyl-2-(phenylethynyl)-pyridine, a mGluR5 antagonist, reinstated the phosphorylation levels of Thr75- and Thr34-DARPP-32, GluR1, and NR1 to control values, and a subsequent cocaine challenge did not elicit a sensitized response. These data suggest that a tonic increase in mGluR5 transmission in cocaine-sensitized rats sustains both the increase in phospho-Thr75 DARPP-32 levels and the expression of behavioral sensitization. PMID:17680995

  11. Ion-exclusion chromatography with conductimetric detection of aliphatic carboxylic acids on a weakly acidic cation-exchange resin by elution with benzoic acid-beta-cyclodextrin.

    PubMed

    Tanaka, Kazuhiko; Mori, Masanobu; Xu, Qun; Helaleh, Murad I H; Ikedo, Mikaru; Taoda, Hiroshi; Hu, Wenzhi; Hasebe, Kiyoshi; Fritz, James S; Haddad, Paul R

    2003-05-16

    In this study, an aqueous solution consisting of benzoic acid with low background conductivity and beta-cyclodextrin (beta-CD) of hydrophilic nature and the inclusion effect to benzoic acid were used as eluent for the ion-exclusion chromatographic separation of aliphatic carboxylic acids with different pKa values and hydrophobicity on a polymethacrylate-based weakly acidic cation-exchange resin in the H+ form. With increasing concentration of beta-cyclodextrin in the eluent, the retention times of the carboxylic acids decreased due to the increased hydrophilicity of the polymethacrylate-based cation-exchange resin surface from the adsorption of OH groups of beta-cyclodextrin. Moreover, the eluent background conductivity decreased with increasing concentration of beta-cyclodextrin in 1 mM benzoic acid, which could result in higher sensitivity for conductimetric detection. The ion-exclusion chromatographic separation of carboxylic acids with high resolution and sensitivity was accomplished successfully by elution with a 1 mM benzoic acid-10 mM cyclodextrin solution without chemical suppression. PMID:12830884

  12. Acidity of the amidoxime functional group in aqueous solution: a combined experimental and computational study.

    PubMed

    Mehio, Nada; Lashely, Mark A; Nugent, Joseph W; Tucker, Lyndsay; Correia, Bruna; Do-Thanh, Chi-Linh; Dai, Sheng; Hancock, Robert D; Bryantsev, Vyacheslav S

    2015-02-26

    Poly(acrylamidoxime) adsorbents are often invoked in discussions of mining uranium from seawater. 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 pK(a) values that have been reported for the amidoxime functional group. To resolve this existing controversy we investigated the pK(a) values of the amidoxime functional group using a combination of experimental and computational methods. Experimentally, we used spectroscopic titrations to measure the pK(a) values of representative amidoximes, acetamidoxime, and benzamidoxime. Computationally, we report on the performance of several protocols for predicting the pK(a) 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 root-mean-square deviation of 0.46 pK(a) units and 0.45 pK(a) units, respectively. Finally, we employ our two best methods to predict the pK(a) values of promising, uncharacterized amidoxime ligands, which provides a convenient means for screening suitable amidoxime monomers for future generations of poly(acrylamidoxime) adsorbents. PMID:25621618

  13. 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. PMID:27105888

  14. Synthesis, Theoretical Analysis, and Experimental pKa Determination of a Fluorescent, Nonsymmetric, In-Out Proton Sponge.

    PubMed

    Belding, Lee; Stoyanov, Peter; Dudding, Travis

    2016-01-01

    Herein, we report the synthesis and theoretical investigation of a nonsymmetric bis(diisopropylamino)cyclopropenimine (DAC)-functionalized proton sponge derivative, coined the "Janus" sponge. The reported sponge was isolated as a monoprotonated salt, though no intramolecular hydrogen bond was observed. Homodesmotic equations supported the absence of a N-HN intramolecular hydrogen bond and a relatively low freebase strain, while DFT calculations and X-ray crystallography revealed the presence of a hydrogen bond to the Cl(-) counterion. Associated with this fact was the rare in-out geometry of the basic nitrogens, which represents the first such instance in a proton sponge not having an ortho-substituent and/or being in a protonated state. Furthermore, NLP donation into the cyclopropenium cation was found to stabilize this unprecedented in-out geometry. The measured pKa was determined to be 23.8, in good agreement with the computed value of 23.9. Lastly, the Janus sponge was found to have fluorescent properties both in the solid state and in solution, which notably represents the first example of a cyclopropenimine-based fluorescent organic compound. PMID:26440446

  15. Effect of chlorine as substituent on the photochemistry and acid-base properties of beta-carboline alkaloids.

    PubMed

    Tarzi, Olga I; Erra-Balsells, Rosa

    2006-02-01

    The UV-absorption, fluorescence excitation and emission spectra of the 6-chloro-, 8-chloro-, 6,8-dichloro-derivatives of nor-harmane, harmane and harmine and the 8-chloro-derivative of harmol were studied. These studies were performed in EtOH and in EtOH+1% perchloric acid solutions (pa). Furthermore, fluorescence quantum yields (phi(f)) in both media and in acetonitrile and acetonitrile + 1% perchloric acid solutions at 298 K were measured. The HOMO and LUMO energy, the positions (lambda(max)) and oscillator strength (f) of the 1S1 <-- 1S0 band for all the neutral and protonated beta-carbolines studied were calculated and compared with the experimental data. The pK(a) values in aqueous solution for for 6-chloro-, 8-chloro- and 6,8-dichloro-nor-harmane, harmane and harmine and 8-chloro-harmol were spectrophotometrically measured (pK(a(H2O)). The change of the acid-base character of these compounds on going from the ground state (pK(a)) to the first electronic excited singlet state (pKa*) as DeltapKa = pKa*-pKa, in ethanol solution at 298 K were calculated (DeltapK(a(EtOH))). Ground-state proton affinity (PA) for all the compounds studied defined as minus the enthalpy change of the reaction M + H(+) --> MH+ (gas state) were calculated. Basicity relative to pyridine (DeltaH(rPy)) defined as the enthalpy change of the isodesmic reaction MH(+) + Py --> M + PyH+ in gas state and in water solution, were also calculated (ab initio calculations). The effect of chlorine as substituent on the photochemistry and acid-base properties of the beta-carboline alkaloids is discussed. PMID:16263305

  16. 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 ...

  17. Predictive value of serum uric acid on left atrial spontaneous echo contrast in non-valvular atrial fibrillation patients

    PubMed Central

    Liao, Hong-Tao; Liu, Fang-Zhou; Xue, Yu-Mei; Zhan, Xian-Zhang; Fang, Xian-Hong; Huang, Jun; Wei, Wei; Rao, Fang; Deng, Hai; Liu, Yang; Lin, Wei-Dong; Wu, Shu-Lin

    2015-01-01

    Objectives To investigate the relationship between serum uric acid (SUA) and left atrial spontaneous echo contrast (LA-SEC) in non-valvular atrial fibrillation (AF) patients. Methods We retrospectively screened 1,476 consecutive hospitalized patients with AF who underwent transesophageal echocardiography prior to radiofrequency catheter ablation, left atrial appendage closure and electric cardioversion at Guangdong General Hospital. Data on the clinical baseline characteristics of all patients were collected from electronic medical records and analyzed. Results After exclusion of patients with left atrial thrombus, 1,354 patients entered into present study and 57 were LA-SEC. The mean female SUA level (380.88 ± 94.35 µmol/L vs. 323.37 ± 72.19 µmol/L, P < 0.001) and male SUA level (416.97 ± 98.87 µmol/L vs. 367.88 ± 68.50 µmol/L, P = 0.008) were both significantly higher in patients with LA-SEC than in the controls. The mean left atrial dimension (41.32 ± 5.12 mm vs. 36.12 ± 5.66 mm, P < 0.001) was markedly larger in patients with LA-SEC. In multivariate regression analysis, SUA level was an independent risk factor for LA-SEC (OR: 1.008, P < 0.001). In receiver operating characteristic curve analysis, the corresponding area under the curve for SUA predicting LA-SEC in female and male were 0.670 and 0.657, respectively. SUA level is significantly higher in non-valvular AF patients with LA-SEC. Conclusion SUA level is an independent risk factor and has a moderate predictive value for LA-SEC among non-valvular AF patients in Southern China. PMID:26788041

  18. 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

  19. c-AMP dependent protein kinase A inhibitory activity of six algal extracts from southeastern Australia and their fatty acid composition.

    PubMed

    Zivanovic, Ana; Skropeta, Danielle

    2012-07-01

    c-AMP dependent protein kinase (protein kinase A, PKA) is an important enzyme involved in the regulation of an increasing number of physiological processes including immune function, cardiovascular disease, memory disorders and cancer. The objective of this study was to evaluate the PKA inhibitory activity of a range of algal extracts, along with their fatty acid composition. Six algal species were investigated including two Chlorophyta (Codium dimorphum and Ulva lactuca), two Phaeophyta (Phyllospora comosa and Sargassum sp.) and two Rhodophyta (Prionitis linearis and Corallina vancouveriensis), with the order of PKA inhibitory activity of their extracts identified as follows: brown seaweeds > red seaweeds > green seaweeds with the brown alga Sargassum sp. exhibiting the highest PKA inhibitory activity (84% at 100 microg/mL). GC/MS analysis identified a total of 18 fatty acids in the six algal extracts accounting for 72-87% of each extract, with hexadecanoic acid and 9,12-octadecadienoic acid as the dominant components. The most active extract (Sargassum sp.) also contained the highest percentage of the saturated C14:0 fatty acid (12.8% of the total extract), which is a known to inhibit PKA. These results provide the first description of the PKA inhibitory activity of marine algae along with the first description of the fatty acid composition of these six algal species from South Eastern Australian waters. Importantly, this study reveals that abundant and readily available marine algae are a new and relatively unexplored source of PKA inhibitory compounds. PMID:22908583

  20. 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

  1. Fluxionate Lewis acidity of the Zn2+ ion in carboxypeptidase A.

    PubMed Central

    Mock, W L; Freeman, D J; Aksamawati, M

    1993-01-01

    Competitive inhibition constants Ki for a series of phenol-ring-substituted derivatives of alpha-(2-hydroxyphenyl)benzenepropanoic acid have been ascertained by observing their influence on the catalytic hydrolysis of a peptide substrate by the zinc enzyme carboxypeptidase A. The pH-dependence of Ki shows that binding is maximal between two pKa values: one is that of the phenol group of the inhibitor, and the other uniformly has a value of 6, the pKa of a Zn(2+)-bound water molecule on the enzyme in the absence of substrate or inhibitor. This is the dependence expected if phenolate binds to the Zn2+ displacing its bound H2O/HO-. A log-log plot of the dissociation constants for the productive forms of inhibitor plus enzyme versus the acid dissociation constants of the phenolic residues in the inhibitors yields a straight line with a slope of +0.76. This number indicates that the active-site metal ion has special capacity for dispersing negative charge, such as builds up on the oxygen atom of a carboxamide group undergoing nucleophilic addition. PMID:8424757

  2. cAMP/PKA signaling inhibits osteogenic differentiation and bone formation in rodent models.

    PubMed

    Siddappa, Ramakrishnaiah; Mulder, Winfried; Steeghs, Ilse; van de Klundert, Christine; Fernandes, Hugo; Liu, Jun; Arends, Roel; van Blitterswijk, Clemens; de Boer, Jan

    2009-08-01

    We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable animal models and cell lines are desirable. In this report, we assessed whether PKA plays a similar proosteogenic role played by two commonly used PKA activators-N6,2'-O-dibutyryl-cAMP (db-cAMP) and 8-bromo cAMP (8b-cAMP)-in a number of model systems. To this end, we treated MC3T3-E1 cells, mouse calvarial osteoblasts, mouse MSCs, and rat MSCs with cAMP. We demonstrate that cAMP inhibits osteogenesis in rodent cell types, evidenced by inhibition of osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (BGLAP), and collagen type 1 (COL1A1). In support of this, ex vivo-cultured mouse calvaria exposed to db-cAMP showed a reduction in bone volume. Interestingly, cAMP even stimulated adipogenic differentiation in rat MSCs. Taken together, our data demonstrate that cAMP inhibits osteogenesis in vitro and bone formation ex vivo in rodent models in contrast to our earlier findings in hMSCs. The species discrepancy in response to various osteogenic signals is a critical need to be tested in clinically relevant models to translate the fundamental findings in lower species level to clinical applications. PMID:19231969

  3. Inferring Phytoplankton, Terrestrial Plant and Bacteria Bulk δ¹³C Values from Compound Specific Analyses of Lipids and Fatty Acids.

    PubMed

    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

  4. 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

  5. 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

  6. Exendin-4 improved rat cortical neuron survival under oxygen/glucose deprivation through PKA pathway.

    PubMed

    Wang, M-D; Huang, Y; Zhang, G-P; Mao, L; Xia, Y-P; Mei, Y-W; Hu, B

    2012-12-13

    Previous studies demonstrated that exendin-4 (Ex-4) may possess neurotrophic and neuroprotective functions in ischemia insults, but its mechanism remained unknown. Here, by using real-time PCR and ELISA, we identified the distribution of active GLP-1Rs in the rat primary cortical neurons. After establishment of an in vitro ischemia model by oxygen/glucose deprivation (OGD), neurons were treated with various dosages of Ex-4. The MTT assay showed that the relative survival rate increased with the dosage of Ex-4 ranging from 0.2 to 0.8 μg/ml (P<0.001, vs. OGD group). The apoptosis rate was reduced from (49.47±2.70)% to (14.61±0.81)% after Ex-4 treatment (0.4 μg/ml) 12h after OGD (P<0.001). Moreover, immunofluorescence staining indicated that Ex-4 increased glucose-regulated proteins 78 (GRP78) and reduced C/EBP-homologous protein (CHOP). Western blot analysis demonstrated that, after neurons were treated with Ex-4, GRP78 was up-regulated over time (P<0.01, vs. OGD group), while CHOP levels rose to a peak 8h after OGD and then decreased (P<0.05, vs. OGD group). This effect was changed by both the protein kinase A (PKA) inhibitor H89 (P<0.01, P<0.05, respectively, vs. Ex-4 group) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (P<0.01, P<0.01, respectively, vs. Ex-4 group) but not by the mitogen-activated protein kinase (MAPK) inhibitor U0126. Our study also revealed that, compared with the Ex-4 group, inhibition of the PKA signaling pathway significantly decreased the survival rate of neurons, down-regulated the expression of B-cell lymphoma 2 (Bcl-2) and up-regulated the Bax expression 3h after ODG (P<0.05, P<0.01, respectively), while neither PI3K nor MAPK inhibition exerted such effects. Furthermore, Western blotting exhibited that PKA expression was elevated in the presence or absence of OGD insults (P<0.05). This study indicated that Ex-4 protected neurons against OGD by modulating the unfolded protein response (UPR) through the PKA pathway and

  7. 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. PMID:25455490

  8. Bile acids and pH values in total feces and in fecal water from habitually omnivorous and vegetarian subjects.

    PubMed

    van Faassen, A; Hazen, M J; van den Brandt, P A; van den Bogaard, A E; Hermus, R J; Janknegt, R A

    1993-12-01

    Twenty habitually omnivorous subjects and 19 habitually lactoovovegetarian subjects aged 59-65 y collected feces during 4 consecutive days. The concentrations of bile acids in total feces did not differ between the omnivores and vegetarians, but the bile acid concentrations in fecal water were significantly lower in the vegetarians. The concentration of the colorectal cancer-predicting bile acid deoxycholic acid in fecal water was explained by the intake of saturated fat and the daily fecal wet weight (r2 = 0.50). Fecal pH did not differ between the omnivores and vegetarians. This variable was significantly (P < 0.05) explained by the intake of calcium (r2 = 0.30); 24-h fecal wet weight and defecation frequency were significantly higher in the vegetarians. In conclusion, our vegetarian subjects had a lower concentration of deoxycholic acid in fecal water, higher fecal wet weight, and higher defecation frequency than the omnivorous subjects. PMID:8249879

  9. Aspects of the chemical stability of mitomycin and porfiromycin in acidic solution.

    PubMed

    Underberg, W J; Lingeman, H

    1983-05-01

    Aspects of the degradations of mitomycin and porfiromycin were studied. The initial degradation processes of the compounds in an acidic medium were investigated. Influences of pH, buffers, and other additives such as halogenides and dioctyl sodium sulfosuccinate [sodium 1,4-bis(2-ethylhexyl)sulfosuccinate] were studied. The hydrogen ion catalyzes the degradation of both the uncharged and the protonated species. Anions also promote the degradation of the compounds in an acidic medium. Rate constants for all of the catalytic reactions could be determined. From the pH profiles, after correction for buffer influences, accurate pKa values for the aziridine nitrogens could be obtained. The protective influence of the dioctyl sulfosuccinate ion could be explained. From the data obtained a plausible mechanism for the initial acidic degradation reactions was developed. PMID:6864504

  10. Acidity of the amidoxime functional group in aqueous solution. A combined experimental and computational study

    DOE PAGESBeta

    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

  11. 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

  12. Comparative redox and pKa calculations on cytochrome c3 from several Desulfovibrio species using continuum electrostatic methods.

    PubMed

    Martel, P J; Soares, C M; Baptista, A M; Fuxreiter, M; Náray-Szabó, G; Louro, R O; Carrondo, M A

    1999-02-01

    A comparative study of the pH-dependent redox mechanisms of several members of the cytochrome c3 family has been carried out. In a previous work, the molecular determinants of this dependency (the so-called redox-Bohr effect) were investigated for one species using continuum electrostatic methods to find groups with a titrating range and strength of interaction compatible with a mediating role in the redox-Bohr effect. Here we clarify these aspects in the light of new and improved pKa calculations, our findings supporting the hypothesis of propionate D from heme I being the main effector in the pH-dependent modulation of the cytochrome c3 redox potentials in all the c3 molecules studied here. However, the weaker (but significant) role of other titrating groups cannot be excluded, their importance and identity changing with the particular molecule under study. We also calculate the relative redox potentials of the four heme centers among the selected members of the c3 family, using a continuum electrostatic method that takes into account both solvation and interaction effects. Comparison of the calculated values with available data for the microscopic redox potentials was undertaken, the quality of the agreement being dependent upon the choice of the dielectric constant for the protein interior. We find that high dielectric constants give best correlations, while low values result in better magnitudes for the calculated potentials. The possibility that the crystallographic calcium ion in c3 from Desulfovibrio gigas may be present in the solution structure was tested, and found to be likely. PMID:10499105

  13. 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

  14. 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

  15. Epac, not PKA catalytic subunit, is required for 3T3-L1 preadipocyte differentiation.

    PubMed

    Ji, Zhenyu; Mei, Fang C; Cheng, Xiaodong

    2010-01-01

    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

  16. Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA.

    PubMed

    Chappe, V; Hinkson, D A; Zhu, T; Chang, X-B; Riordan, J R; Hanrahan, J W

    2003-04-01

    Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the '9CA' mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5-10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wild-type channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by PKC and for its modulation of CFTR responses to PKA. PMID:12588899

  17. Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA

    PubMed Central

    Chappe, V; Hinkson, D A; Zhu, T; Chang, X-B; Riordan, J R; Hanrahan, J W

    2003-01-01

    Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the ‘9CA’ mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5–10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wild-type channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by PKC and for its modulation of CFTR responses to PKA. PMID:12588899

  18. 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

  19. Current evidence for the diagnostic value of gadoxetic acid-enhanced magnetic resonance imaging for liver metastasis.

    PubMed

    Tsurusaki, Masakatsu; Sofue, Keitaro; Murakami, Takamichi

    2016-08-01

    A variety of imaging techniques, including ultrasonography (US), multidetector computed tomography (MDCT), magnetic resonance imaging (MRI) and positron emission tomography combined with CT scan (PET/CT), are available for diagnosis and treatment planning in liver metastasis. Contrast-enhanced MDCT is a relatively non-invasive, widely available and standardized method for hepatic work-up. Gadoxetic acid (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid; EOB Primovist®]) is a recently developed liver-specific hepatobiliary MR contrast agent that offers both dynamic imaging as well as liver-specific static hepatocyte imaging, referred to as the hepatobiliary phase. Following contrast injection, this technique reveals dynamic vascular phases (arterial, portal venous and delayed phases), in addition to the hepatobiliary phase upon uptake by functional hepatocytes. The overall sensitivity of gadoxetic acid-enhanced MRI was significantly higher than that of contrast-enhanced CT. Specifically, the higher sensitivity of gadoxetic acid-enhanced MRI was observed in lesions smaller than 1 cm in diameter. Gadoxetic acid-enhanced MRI is considered an extremely useful tool for the diagnosis of liver metastases. Future studies will focus on diagnostic algorithms involving combinations of modalities such as MRI, MDCT and/or (18) F-fluorodeoxyglucose PET/CT, which may impact the treatment plan for these patients. PMID:26750497

  20. 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

  1. Activation of adenosine A2A receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation

    PubMed Central

    Mediero, Aránzazu; Perez-Aso, Miguel; Cronstein, Bruce N

    2013-01-01

    Background and Purpose We previously reported that adenosine, acting at adenosine A2A receptors (A2AR), inhibits osteoclast (OC) differentiation in vitro (A2AR activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A2AR stimulation regulates OC differentiation, we dissected the signalling pathways involved in A2AR signalling. Experimental Approach OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A2AR agonist CGS21680, the A2AR antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR. Key Results A2AR stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A2AR stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A2AR stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A2AR stimulation inhibited OC expression of differentiation markers by a PKA-mechanism. Conclusions and Implications A2AR activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like Rheumatoid Arthritis. PMID:23647065

  2. Superacidity of closo-dodecaborate-based Brønsted acids: a DFT study.

    PubMed

    Lipping, Lauri; Leito, Ivo; Koppel, Ivar; Krossing, Ingo; Himmel, Daniel; Koppel, Ilmar A

    2015-01-29

    The structures and intrinsic gas-phase acidities (GA) of some dodecaborane acids, the derivatives of YB12H11H (Y = PF3, NH3, NF3, NMe3), B12H12H2, and B12H12H(-) (HA, H2A, and HA(-), respectively) have been computationally explored with DFT B3LYP method at the 6-311+G** level of theory as new possible directions of creating superstrong Brønsted acids. Depending on the nature and number of the substituents different protonation geometries were investigated. In general, the GA values of the neutral systems varied according to the substituents in the following order: CF3 < F < Cl and in case of anionic acids: CF3 < Cl < F. The dodecatrifluoromethyl derivative of H2A, B12(CF3)12H1H2, emerges as the strongest among the considered acids and is expected to be in the gas phase at least as strong as the undecatrifluoromethyl carborane, CB11(CF3)11H1H. The GA values of the respective monoanionic forms of the considered acids all, but the (CF3)11 derivative, remained higher than the widely used threshold of superacidity. The HA derivatives' (Y = PF3, NF3) GA's were approximately in the same range as the H2A acids'. In the case Y = NH3 or NMe3 the GA values were significantly higher. Also, the pKa values of B12H12H2, CB11H12H, and their perfluorinated derivatives in 1,2-dichloroethane (DCE) were estimated with SMD and cluster-continuum model calculations. The obtained estimates of pKa values of the perfluorinated derivatives are by around 30 units lower than that of trifluoromethylsulfonylimide, making these acids the strongest ever predicted in solution. The derivatives of B12H12H2 are as a rule not significantly weaker acids than the respective derivatives of CB11H12H. This is important for expanding practical applicability of this type of acids and their anions, as they are synthetically much more accessible than the corresponding CB11H12(-) derivatives. PMID:25513897

  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. Glucagon Like Peptide-1 (GLP-1) Modulates OVA-Induced Airway Inflammation and Mucus Secretion Involving a Protein Kinase A (PKA)-Dependent Nuclear Factor-κB (NF-κB) Signaling Pathway in Mice

    PubMed Central

    Zhu, Tao; Wu, Xiao-ling; Zhang, Wei; Xiao, Min

    2015-01-01

    Asthma is a common chronic pulmonary inflammatory disease, featured with mucus hyper-secretion in the airway. Recent studies found that glucagon like peptide-1 (GLP-1) analogs, including liraglutide and exenatide, possessed a potent anti-inflammatory property through a protein kinase A (PKA)-dependent signaling pathway. Therefore, the aim of current study was to investigate the value of GLP-1 analog therapy liraglutide in airway inflammation and mucus secretion in a murine model of ovalbumin (OVA)-induced asthma, and its underlying molecular mechanism. In our study, BALB/c mice were sensitized and challenged by OVA to induce chronic asthma. Pathological alterations, the number of cells and the content of inflammatory mediators in bronchoalveolar lavage fluid (BALF), and mucus secretion were observed and measured. In addition, the mRNA and protein expression of E-selectin and MUC5AC were analyzed by qPCR and Western blotting. Then, the phosphorylation of PKA and nuclear factor-κB (NF-κB) p65 were also measured by Western blotting. Further, NF-κB p65 DNA binding activity was detected by ELISA. OVA-induced airway inflammation, airway mucus hyper-secretion, the up-regulation of E-selectin and MUC5AC were remarkably inhibited by GLP-1 in mice (all p < 0.01). Then, we also found that OVA-reduced phosphorylation of PKA, and OVA-enhanced NF-κB p65 activation and NF-κB p65 DNA binding activity were markedly improved by GLP-1 (all p < 0.01). Furthermore, our data also figured out that these effects of GLP-1 were largely abrogated by the PKA inhibitor H-89 (all p < 0.01). Taken together, our results suggest that OVA-induced asthma were potently ameliorated by GLP-1 possibly through a PKA-dependent inactivation of NF-κB in mice, indicating that GLP-1 analogs may be considered an effective and safe drug for the potential treatment of asthma in the future. PMID:26343632

  5. Thyrotropin but not epidermal growth factor down-regulates the isozyme I (PKa I) of cyclic AMP-dependent protein kinases in dog thyroid cells in primary cultures.

    PubMed

    Breton, M F; Roger, P P; Omri, B; Dumont, J E; Pavlovic-Hournac, M

    1989-01-01

    The activity of the two cAMP-dependent protein kinases (PKa I and PKa II) was evaluated in dog thyroid cells in primary cultures after a 6-day growth period induced by either thyrotropin (TSH) or epidermal growth factor (EGF). Although the total PKa activity was not affected in cells cultured in the presence of TSH or EGF, their actions on the PKa I and PKa II expressions were significantly different. The activity of PKa I was strongly inhibited by TSH (70-80%) while with EGF it was either stimulated or unaffected with respect to controls. The two mitogens did not have a significant effect on the activity of PKa II. Forskolin (Fk) mimicked the effect of TSH. The expression of the two regulatory subunits (R I and R II), evaluated by the covalent binding of 8-azido-cAMP, was similar to the expression of the corresponding catalytic activities, suggesting a coregulation of the catalytic and regulatory subunits from the same isozyme. After chronic stimulation by TSH, differentiated dog thyroid cells are almost completely deprived of PKa I. PMID:2545480

  6. 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...

  7. 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…

  8. 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.

  9. Dynamics of PKA phosphorylation and gain of function in cardiac pacemaker cells: a computational model analysis.

    PubMed

    Behar, Joachim; Yaniv, Yael

    2016-05-01

    Cardiac pacemaker cell function is regulated by a coupled-clock system that integrates molecular cues on the cell-membrane surface (i.e., membrane clock) and on the sarcoplasmic reticulum (SR) (i.e., Ca(2+) clock). A recent study has shown that cotransfection of spontaneous beating cells (HEK293 cells and neonatal rat myocytes) with R524Q-mutant human hyperpolarization-activated cyclic nucleotide-gated molecules (the dominant component of funny channels) increases the funny channel's sensitivity to cAMP and leads to a decrease in spontaneous action potential (AP) cycle length (i.e., tachycardia). We hypothesize that in rabbit pacemaker cells, the same behavior is expected, and because of the coupled-clock system, the resultant steady-state decrease in AP cycle length will embody contributions from both clocks: the initial decrease in the spontaneous AP beating interval, arising from increased sensitivity of the f-channel to cAMP, will be accompanied by an increase in the adenylyl cyclase (AC)-cAMP-PKA-dependent phosphorylation activity, which will further decrease this interval. To test our hypothesis, we used the recently developed Yaniv-Lakatta pacemaker cell numerical model. This model predicts the cAMP signaling dynamics, as well as the kinetics and magnitude of protein phosphorylation in both normal and mutant pacemaker cells. We found that R524Q-mutant pacemaker cells have a shorter AP firing rate than that of wild-type cells and that gain in pacemaker function is the net effect of the R514Q mutation on the functioning of the coupled-clock system. Specifically, our results directly support the hypothesis that changes in Ca(2+)-activated AC-cAMP-PKA signaling are involved in the development of tachycardia in R524Q-mutant pacemaker cells. PMID:26945074

  10. Effects of a propionic acid-based preservative on storage characteristics, nutritive value, and energy content for alfalfa hays packaged in large round bales.

    PubMed

    Coblentz, W K; Bertram, M G

    2012-01-01

    During 2009 and 2010, alfalfa (Medicago sativa L.) hays from 2 cuttings harvested from the same field site were used to evaluate the effects of a propionic acid-based preservative on the storage characteristics and nutritive value of hays stored as large round bales. A total of 87 large round bales (diameter = 1.5m) were included in the study; of these, 45 bales served as controls, whereas 42 were treated with a commercial propionic acid-based preservative at mean application rates of 0.5±0.14 and 0.7±0.19% of bale weight, expressed on a wet (as is) or dry matter basis, respectively. Initial bale moisture concentrations ranged from 10.2 to 40.4%. Internal bale temperatures were monitored daily during an outdoor storage period, and heating characteristics were summarized for each bale as heating degree days (HDD) >30°C. For acid-treated bales, the regression relationship between HDD and initial bale moisture was best fitted to a quadratic model in which the linear term was dropped to improve fit (Y=2.02x(2) - 401; R(2)=0.77); control hays were best fitted to a nonlinear model in which the independent variable was squared [Y=4,112 - (4,549×e(-0.000559x*x)); R(2)=0.77]. Based on these regressions, acid-treated bales accumulated more HDD than control hays when the initial bale moisture was >27.7%; this occurred largely because acid treatment tended to prolong active heating relative to control hays. Linear regressions of recoveries of dry matter on HDD did not differ on the basis of treatment, yielding a common linear relationship of Y=-0.0066x+96.3 (R(2)=0.75). Regressions relating changes (post-storage - pre-storage) in concentrations of several nutritional components (neutral detergent fiber, lignin, ash, crude protein, and total digestible nutrients) with HDD for acid-treated hays typically exhibited more inflection points or were higher-ordered polynomial regressions than those of control hays. These more complex responses probably reflected the perturbation

  11. 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

  12. 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

  13. 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

  14. Capillary electrophoresis analysis of organic amines and amino acids in saline and acidic samples using the Mars organic analyzer.

    PubMed

    Stockton, Amanda M; Chiesl, Thomas N; Lowenstein, Tim K; Amashukeli, Xenia; Grunthaner, Frank; Mathies, Richard A

    2009-11-01

    The Mars Organic Analyzer (MOA) has enabled the sensitive detection of amino acid and amine biomarkers in laboratory standards and in a variety of field sample tests. However, the MOA is challenged when samples are extremely acidic and saline or contain polyvalent cations. Here, we have optimized the MOA analysis, sample labeling, and sample dilution buffers to handle such challenging samples more robustly. Higher ionic strength buffer systems with pK(a) values near pH 9 were developed to provide better buffering capacity and salt tolerance. The addition of ethylaminediaminetetraacetic acid (EDTA) ameliorates the negative effects of multivalent cations. The optimized protocol utilizes a 75 mM borate buffer (pH 9.5) for Pacific Blue labeling of amines and amino acids. After labeling, 50 mM (final concentration) EDTA is added to samples containing divalent cations to ameliorate their effects. This optimized protocol was used to successfully analyze amino acids in a saturated brine sample from Saline Valley, California, and a subcritical water extract of a highly acidic sample from the Río Tinto, Spain. This work expands the analytical capabilities of the MOA and increases its sensitivity and robustness for samples from extraterrestrial environments that may exhibit pH and salt extremes as well as metal ions. PMID:19968460

  15. Rhes influences striatal cAMP/PKA-dependent signaling and synaptic plasticity in a gender-sensitive fashion.

    PubMed

    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

  16. 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

  17. Defining A-Kinase Anchoring Protein (AKAP) Specificity for the Protein Kinase A Subunit RI (PKA-RI).

    PubMed

    Autenrieth, Karolin; Bendzunas, N George; Bertinetti, Daniela; Herberg, Friedrich W; Kennedy, Eileen J

    2016-04-15

    A-Kinase anchoring proteins (AKAPs) act as spatial and temporal regulators of protein kinase A (PKA) by localizing PKA along with multiple proteins into discrete signaling complexes. AKAPs interact with the PKA holoenzyme through an α-helix that docks into a groove formed on the dimerization/docking domain of PKA-R in an isoform-dependent fashion. In an effort to understand isoform selectivity at the molecular level, a library of protein-protein interaction (PPI) disruptors was designed to systematically probe the significance of an aromatic residue on the AKAP docking sequence for RI selectivity. The stapled peptide library was designed based on a high affinity, RI-selective disruptor of AKAP binding, RI-STAD-2. Phe, Trp and Leu were all found to maintain RI selectivity, whereas multiple intermediate-sized hydrophobic substitutions at this position either resulted in loss of isoform selectivity (Ile) or a reversal of selectivity (Val). As a limited number of RI-selective sequences are currently known, this study aids in our understanding of isoform selectivity and establishing parameters for discovering additional RI-selective AKAPs. PMID:26611881

  18. 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…

  19. VIP, CRF, and PACAP Act at Distinct Receptors to Elicit Different cAMP/PKA Dynamics in the Neocortex

    PubMed Central

    Hu, Emilie; Demmou, Lynda; Cauli, Bruno; Gallopin, Thierry; Geoffroy, Hélène; Harris-Warrick, Ronald M.; Paupardin-Tritsch, Danièle; Lambolez, Bertrand; Vincent, Pierre

    2011-01-01

    The functional significance of diverse neuropeptide coexpression and convergence onto common second messenger pathways remains unclear. To address this question, we characterized responses to corticotropin-releasing factor (CRF), pituitary adenylate cyclase–activating peptide (PACAP), and vasoactive intestinal peptide (VIP) in rat neocortical slices using optical recordings of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) sensors, patch-clamp, and single-cell reverse transcription–polymerase chain reaction. Responses of pyramidal neurons to the 3 neuropeptides markedly differed in time-course and amplitude. Effects of these neuropeptides on the PKA-sensitive slow afterhyperpolarization current were consistent with those observed with cAMP/PKA sensors. CRF-1 receptors, primarily expressed in pyramidal cells, reportedly mediate the neocortical effects of CRF. PACAP and VIP activated distinct PAC1 and VPAC1 receptors, respectively. Indeed, a selective VPAC1 antagonist prevented VIP responses but had a minor effect on PACAP responses, which were mimicked by a specific PAC1 agonist. While PAC1 and VPAC1 were coexpressed in pyramidal cells, PAC1 expression was also frequently detected in interneurons, suggesting that PACAP has widespread effects on the neuronal network. Our results suggest that VIP and CRF, originating from interneurons, and PACAP, expressed mainly by pyramidal cells, finely tune the excitability and gene expression in the neocortical network via distinct cAMP/PKA-mediated effects. PMID:20699230

  20. Evidence for bias in measured δ15N values of terrestrial and aquatic organic materials due to pre-analysis acid treatment methods.

    PubMed

    Brodie, Chris R; Heaton, Tim H E; Leng, Melanie J; Kendrick, Christopher P; Casford, James S L; Lloyd, Jeremy M

    2011-04-30

    We investigate the effect of acid treatment methods on δ(15)N values from a range of environmental organic materials in the context of the increased application of 'dual-mode' isotope analysis (the simultaneous measurement of δ(13)C and δ(15)N from the same acid-treated sample). Three common methods are compared; (i) untreated samples; (ii) acidification followed by sequential water rinse (rinse method); and (iii) acidification in silver capsules (capsule method). The influence of capsule type (silver and tin) on δ(15)N is also independently assessed (as the capsule and rinse methods combust samples in different capsules; silver and tin, respectively). We find significant differences in δ(15)N values between methods and the precision of any one method varies significantly between sample materials and above the instrument precision (>0.3‰). The δ(15)N values of untreated samples did not produce the most consistent data on all sample materials. In addition, the capsule type appears to influence the measured δ(15)N value of some materials, particularly those combusted only in silver capsules. We also compare the new δ(15)N data with previously published δ(13)C data on the same materials. The response of δ(13)C and δ(15)N within and between methods and sample materials to acidification appears to be relatively disproportionate, which can influence the environmental interpretation of the measured data. In addition, statistical methods used to estimate inorganic nitrogen are shown to be seriously flawed. PMID:21452387

  1. 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.

  2. 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

  3. [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

  4. Estimation of pKB values for histamine H2-receptor antagonists using an in vitro acid secretion assay.

    PubMed Central

    Angus, J. A.; Black, J. W.; Stone, M.

    1980-01-01

    1 Histamine H2-receptor antagonism by burimamide, metiamide and cimetidine was analysed under apparent equilibrium conditions in the lumen-perfused isolated stomach preparation of the mouse. 2 The behaviour of these compounds was not incompatible with simple competitive antagonism but the estimated pKB values (-log KB) were all significantly lower, by about 1 log unit, than reference values reported for guinea-pig atrium or rat uterus. 3 There seems no need to propose that the parietal cell receptors are somehow different from the others; metiamide continually appears in the gastric juice so that a steady-state but not equilibrium can presumably be reached with respect to the bath concentration and this could keep the antagonist concentration artificially low in the region of the receptors. PMID:6132635

  5. Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN' by NMR.

    PubMed

    Day, Regina M; Thalhauser, Craig J; Sudmeier, James L; Vincent, Matthew P; Torchilin, Ekaterina V; Sanford, David G; Bachovchin, Christopher W; Bachovchin, William W

    2003-04-01

    We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02. PMID:12649438

  6. Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN′ by NMR

    PubMed Central

    Day, Regina M.; Thalhauser, Craig J.; Sudmeier, James L.; Vincent, Matthew P.; Torchilin, Ekaterina V.; Sanford, David G.; Bachovchin, Christopher W.; Bachovchin, William W.

    2003-01-01

    We have determined by 15N, 1H, and 13C NMR, the chemical behavior of the six histidines in subtilisin BPN′ and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every 15N, 1H, Cɛ1, and Cδ2 resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pKa = 7.30 ± 0.03 at 25°C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pKa value of 7.9 ± 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high Cɛ1-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved Cɛ1-H. . .O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare Nδ1-H tautomer, exhibiting 13Cδ1 chemical shifts ~9 ppm higher than those for Nɛ2-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by 15N-1H NOE effects, and titrates with rapid proton exchange kinetics linked to a pKa value of 7.47 ± 0.02. PMID:12649438

  7. "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

  8. 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. PMID:26153501

  9. FT-IR, Raman and DFT study of 2-amino-5-fluorobenzoic acid and its biological activity with other halogen (Cl, Br) substitution.

    PubMed

    Xavier, T S; Joe, I Hubert

    2011-07-01

    The Fourier-transform Raman and infrared spectra of 2-amino-5-fluoro benzoic acid has been recorded and analyzed. The optimized geometry of the other halogen substitution (Cl, Br) have been computed with the help of density functional theory. The detailed interpretation of vibrational spectra of 2-amino-5-fluoro benzoic acid have performed in terms of potential energy distribution analysis. Natural bond orbital analysis on 2-amino-5-fluoro benzoic acid, 2-amino-5-chloro benzoic acid and 2-amino-5-bromo benzoic acid has been carried out for various intramolecular interactions that are responsible for the stabilization of the molecule. The pKa values of 2-amino-5-fluoro benzoic acid, 2-amino-5-chloro benzoic acid and 2-amino-5-bromo benzoic acid are computed using MOPAC and it is related with HOMO-LUMO energy difference obtained from Gaussian 03 software. The biological activity of 2-amino-5-fluoro benzoic acid has been predicted based on these values. The inhibition activity of 2-amino-5-bromo benzoic acid with the protein tyrosine kinase 3LQ8 is simulated by using Autodock software. PMID:21497545

  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. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.

    PubMed

    Noble, M A; Gul, S; Verma, C S; Brocklehurst, K

    2000-11-01

    The pK(a) of (Asp(158))-CO(2)H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys(25)/His(159) nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state comprising (Cys(25))-S(-)/(His(159))-Im(+)H (in which Im represents imidazole) produced by protonic dissociation of (Cys(25))-SH/(His(159))-Im(+)H with pK(a) 3.3 and (Asp(158))-CO(2)H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH-k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pK(a) value of (Asp(158))-CO(2)H of caricain, which was calculated to be 2.0 by solving the linearized Poisson-Boltzmann equation with the program UHBD ('University of Houston Brownian dynamics'). A value lower than 2.8 had been predicted by consideration of the hydrogen-bonded networks involving Asp(158) and its microenvironments in both enzymes. The difference between these pK(a) values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp(158))-CO(2)(-), which modifies traditional mechanistic views, is discussed. PMID:11042128

  12. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry.

    PubMed Central

    Noble, M A; Gul, S; Verma, C S; Brocklehurst, K

    2000-01-01

    The pK(a) of (Asp(158))-CO(2)H of papain (EC 3.4.22.2) was determined as 2.8 by using 4-chloro-7-nitrobenzofurazan (Nbf-Cl) as a reactivity probe targeted on the thiolate anion component of the Cys(25)/His(159) nucleophilic-acid/base motif of the catalytic site. The possibility of using Nbf-Cl for this purpose was established by modelling the papain-Nbf-Cl Meisenheimer intermediate by using QUANTA/CHARMM and performing molecular orbital calculations with MOPAC interfaced with Cerius 2. A pH-dependent stopped-flow kinetic study of the reaction of papain with Nbf-Cl established that the striking rate maximum at pH 3 results from reaction in a minor ionization state comprising (Cys(25))-S(-)/(His(159))-Im(+)H (in which Im represents imidazole) produced by protonic dissociation of (Cys(25))-SH/(His(159))-Im(+)H with pK(a) 3.3 and (Asp(158))-CO(2)H. Although the analogous intermediate in the reaction of caricain (EC 3.4.22.30) with Nbf-Cl has similar geometry, the pH-k profile (k being the second-order rate constant) lacks a rate maximum under acidic conditions. This precludes the experimental determination of the pK(a) value of (Asp(158))-CO(2)H of caricain, which was calculated to be 2.0 by solving the linearized Poisson-Boltzmann equation with the program UHBD ('University of Houston Brownian dynamics'). A value lower than 2.8 had been predicted by consideration of the hydrogen-bonded networks involving Asp(158) and its microenvironments in both enzymes. The difference between these pK(a) values (values not previously detected in reactions of either enzyme) accounts for the lack of the rate maximum in the caricain reaction and for the differences in the electronic absorption spectra of the two S-Nbf-enzymes under acidic conditions. The concept of control of cysteine proteinase activity by multiple electrostatic modulators, including (Asp(158))-CO(2)(-), which modifies traditional mechanistic views, is discussed. PMID:11042128

  13. 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

  14. 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.

  15. Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity.

    PubMed

    Nader, Nancy; Courjaret, Raphael; Dib, Maya; Kulkarni, Rashmi P; Machaca, Khaled

    2016-06-01

    Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway. PMID:27122173

  16. 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.

  17. 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

  18. 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

  19. Role of PKA signaling in D2 receptor-expressing neurons in the core of the nucleus accumbens in aversive learning

    PubMed Central

    Yamaguchi, Takashi; Goto, Akihiro; Nakahara, Ichiro; Yawata, Satoshi; Hikida, Takatoshi; Matsuda, Michiyuki; Funabiki, Kazuo; Nakanishi, Shigetada

    2015-01-01

    The nucleus accumbens (NAc) serves as a key neural substrate for aversive learning and consists of two distinct subpopulations of medium-sized spiny neurons (MSNs). The MSNs of the direct pathway (dMSNs) and the indirect pathway (iMSNs) predominantly express dopamine (DA) D1 and D2 receptors, respectively, and are positively and negatively modulated by DA transmitters via Gs- and Gi-coupled cAMP-dependent protein kinase A (PKA) signaling cascades, respectively. In this investigation, we addressed how intracellular PKA signaling is involved in aversive learning in a cell type-specific manner. When the transmission of either dMSNs or iMSNs was unilaterally blocked by pathway-specific expression of transmission-blocking tetanus toxin, infusion of PKA inhibitors into the intact side of the NAc core abolished passive avoidance learning toward an electric shock in the indirect pathway-blocked mice, but not in the direct pathway-blocked mice. We then examined temporal changes in PKA activity in dMSNs and iMSNs in behaving mice by monitoring Förster resonance energy transfer responses of the PKA biosensor with the aid of microendoscopy. PKA activity was increased in iMSNs and decreased in dMSNs in both aversive memory formation and retrieval. Importantly, the increased PKA activity in iMSNs disappeared when aversive memory was prevented by keeping mice in the conditioning apparatus. Furthermore, the increase in PKA activity in iMSNs by aversive stimuli reflected facilitation of aversive memory retention. These results indicate that PKA signaling in iMSNs plays a critical role in both aversive memory formation and retention. PMID:26305972

  20. The Role of Organic Acids in the Acid-Base Status of Surface Waters at Bickford Watershed, Massachusetts

    NASA Astrophysics Data System (ADS)

    Eshleman, K. N.; Hemond, H. F.

    1985-10-01

    An experimental field study of the alkalinity and major ion budgets of Bickford watershed in central Massachusetts indicates that organic acid production by the ecosystem contributes measurably to surface water acidification. Applying the concepts of alkalinity, electroneutrality of solutions, and mass balance, organic acids were found to comprise 20% of all strong acid sources on one subcatchment annually, a value half as large as the measured bulk mineral acid deposition. Inorganic cation to anion ratios in Provencial Brook varied between 1.0 in winter and 1.6 during summer, suggesting the presence of up to 100 μeq/L of unmeasured charge from organic anions during the growing season. Base titrations and ultraviolet photooxidation experiments confirmed the existence of low pKa (3.5-5.0) acidic functional groups. A positive linear relationship between dissolved organic carbon (DOC) and anion deficit for a group of surface and groundwater samples indicates the DOC contains about 7.5 meq carboxylic groups per gram C. Biological factors related to both upland and wetland carbon metabolism apparently control this natural acidification phenomenon, which has not been documented on other watersheds in the northeastern United States for which annual alkalinity budgets have been determined.

  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. Uncovering the pKa dependent fluorescence quenching of carbon dots induced by chlorophenols

    NASA Astrophysics Data System (ADS)

    Zhang, Yu; Wang, Yu; Guan, Yafeng; Feng, Liang

    2015-03-01

    Fluorescence quenching induced by targets is always an alluring strategy to elucidate the possible photoluminescence origin of carbon dots. In this study, a new kind of N, S co-doped carbon dots (NSCDs) was synthesized and the fluorescence of NSCDs was surprisingly found to be quenched by chlorophenols (CPs) in a pKa dependent mode. Detailed investigation of this behavior demonstrated that phenolate was the responsible species and N and/or S dopants in NSCDs failed to play a role in the fluorescence quenching. Further evidence uncovered that the quenching was a static one, where a non-fluorescent intermediate was formed between electron-deficient C&z.dbd;O on the CDs surface and the electron-rich phenolic oxygen anion of chlorophenolate via nucleophilic addition. Moreover, one of the main photoluminescence origins of this kind of CDs was derived, namely surface emissive sites mostly attributed to carbonyl groups.Fluorescence quenching induced by targets is always an alluring strategy to elucidate the possible photoluminescence origin of carbon dots. In this study, a new kind of N, S co-doped carbon dots (NSCDs) was synthesized and the fluorescence of NSCDs was surprisingly found to be quenched by chlorophenols (CPs) in a pKa dependent mode. Detailed investigation of this behavior demonstrated that phenolate was the responsible species and N and/or S dopants in NSCDs failed to play a role in the fluorescence quenching. Further evidence uncovered that the quenching was a static one, where a non-fluorescent intermediate was formed between electron-deficient C&z.dbd;O on the CDs surface and the electron-rich phenolic oxygen anion of chlorophenolate via nucleophilic addition. Moreover, one of the main photoluminescence origins of this kind of CDs was derived, namely surface emissive sites mostly attributed to carbonyl groups. Electronic supplementary information (ESI) available: Texts, figures and tables giving partial experimental procedures, detailed characterizations

  3. Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII

    PubMed Central

    Fischer, Thomas H.; Herting, Jonas; Mason, Fleur E.; Hartmann, Nico; Watanabe, Saera; Nikolaev, Viacheslav O.; Sprenger, Julia U.; Fan, Peidong; Yao, Lina; Popov, Aron-Frederik; Danner, Bernhard C.; Schöndube, Friedrich; Belardinelli, Luiz; Hasenfuss, Gerd; Maier, Lars S.; Sossalla, Samuel

    2015-01-01

    Aims Enhanced cardiac late Na current (late INa) and increased sarcoplasmic reticulum (SR)-Ca2+-leak are both highly arrhythmogenic. This study seeks to identify signalling pathways interconnecting late INa and SR-Ca2+-leak in atrial cardiomyocytes (CMs). Methods and results In murine atrial CMs, SR-Ca2+-leak was increased by the late INa enhancer Anemonia sulcata toxin II (ATX-II). An inhibition of Ca2+/calmodulin-dependent protein kinase II (Autocamide-2-related inhibitory peptide), protein kinase A (H89), or late INa (Ranolazine or Tetrodotoxin) all prevented ATX-II-dependent SR-Ca2+-leak. The SR-Ca2+-leak induction by ATX-II was not detected when either the Na+/Ca2+ exchanger was inhibited (KBR) or in CaMKIIδc-knockout mice. FRET measurements revealed increased cAMP levels upon ATX-II stimulation, which could be prevented by inhibition of adenylyl cyclases (ACs) 5 and 6 (NKY 80) but not by inhibition of phosphodiesterases (IBMX), suggesting PKA activation via an AC-dependent increase of cAMP levels. Western blots showed late INa-dependent hyperphosphorylation of CaMKII as well as PKA target sites at ryanodine receptor type-2 (-S2814 and -S2808) and phospholamban (-Thr17, -S16). Enhancement of late INa did not alter Ca2+-transient amplitude or SR-Ca2+-load. However, upon late INa activation and simultaneous CaMKII inhibition, Ca2+-transient amplitude and SR-Ca2+-load were increased, whereas PKA inhibition reduced Ca2+-transient amplitude and load and additionally slowed Ca2+ elimination. In atrial CMs from patients with atrial fibrillation, inhibition of late INa, CaMKII, or PKA reduced the SR-Ca2+-leak. Conclusion Late INa exerts distinct effects on Ca2+ homeostasis in atrial myocardium through activation of CaMKII and PKA. Inhibition of late INa represents a potential approach to attenuate CaMKII activation and decreases SR-Ca2+-leak in atrial rhythm disorders. The interconnection with the cAMP/PKA system further increases the antiarrhythmic potential of late

  4. 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. PMID:23595830

  5. Gamma-hydroxybutyric acid endogenous production and post-mortem behaviour - the importance of different biological matrices, cut-off reference values, sample collection and storage conditions.

    PubMed

    Castro, André L; Dias, Mário; Reis, Flávio; Teixeira, Helena M

    2014-10-01

    Gamma-Hydroxybutyric Acid (GHB) is an endogenous compound with a story of clinical use, since the 1960's. However, due to its secondary effects, it has become a controlled substance, entering the illicit market for recreational and "dance club scene" use, muscle enhancement purposes and drug-facilitated sexual assaults. Its endogenous context can bring some difficulties when interpreting, in a forensic context, the analytical values achieved in biological samples. This manuscript reviewed several crucial aspects related to GHB forensic toxicology evaluation, such as its post-mortem behaviour in biological samples; endogenous production values, whether in in vivo and in post-mortem samples; sampling and storage conditions (including stability tests); and cut-off reference values evaluation for different biological samples, such as whole blood, plasma, serum, urine, saliva, bile, vitreous humour and hair. This revision highlights the need of specific sampling care, storage conditions, and cut-off reference values interpretation in different biological samples, essential for proper practical application in forensic toxicology. PMID:25287794

  6. Acido-basic properties of proton pump inhibitors in aqueous solutions.

    PubMed

    Kristl, Albin

    2009-01-01

    The pharmacological characteristics of proton pump inhibitors are related to their protolytic behavior estimated by their pK(a) values. Lansoprazole is a potent anti-acid drug from this group. Because of its poor stability a rapid spectrophotometric method was developed for the determination of its pK(a) values. Three pK(a) values were obtained: an acidic pK(a1) = 8.84 and two basic, pK(a2) = 4.15 and pK(a3) = 1.33. These pK(a) values were discussed from the point of lansoprazole structure and instability with the aim of locating basic and acidic moieties in the molecule of proton pump inhibitors. They were also compared with experimentally determined pK(a) values from the literature and with some pK(a) values calculated by different programs. PMID:18720145

  7. Conversion of distiller's grain into fuel alcohol and a higher-value animal feed by dilute-acid pretreatment.

    PubMed

    Tucker, Melvin P; Nagle, Nicholas J; Jennings, Edward W; Ibsen, Kelly N; Aden, Andy; Nguyen, Quang A; Kim, Kyoung H; Noll, Sally L

    2004-01-01

    Over the past three decades ethanol production in the United States has increased more than 10-fold, to approx 2.9 billion gal/yr (mid-2003), with ethanol production expected to reach 5 billion gal/yr by 2005. The simultaneous coproduction of 7 million t/yr of distiller's grain (DG) may potentially drive down the price of DG as a cattle feed supplement. The sale of residual DG for animal feed is an important part of corn dry-grind ethanol production economics; therefore, dry-grind ethanol producers are seeking ways to improve the quality of DG to increase market penetration and help stabilize prices. One possible improvement is to increase the protein content of DG by converting the residual starch and fiber into ethanol. We have developed methods for steam explosion, SO2, and dilute-sulfuric acid pretreatment of DG for evaluation as a feedstock for ethanol production. The highest soluble sugar yields (approximately 77% of available carbohydrate) were obtained by pretreatment of DG at 140 degrees C for 20 min with 3.27 wt% H2SO4. Fermentation protocols for pretreated DG were developed at the bench scale and scaled to a working volume of 809 L for production of hydrolyzed distiller's grain (HDG) for feeding trials. The pretreated DG was fermented with Saccharomyces cerevisiae D5A, with ethanol yields of 73% of theoretical from available glucans. The HDG was air-dried and used for turkey-feeding trials. The inclusion of HDG into turkey poult (as a model non-ruminant animal) diets at 5 and 10% levels, replacing corn and soybean meal, showed weight gains in the birds similar to controls, whereas 15 and 20% inclusion levels showed slight decreases (-6%) in weight gain. At the conclusion of the trial, no negative effects on internal organs or morphology, and no mortality among the poults, was found. The high protein levels (58-61%) available in HDG show promising economics for incorporation of this process into corn dry-grind ethanol plants. PMID:15054259

  8. 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. PMID:25952875

  9. 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.

  10. Confinement Sensing and Signal Optimization via Piezo1/PKA and Myosin II Pathways.

    PubMed

    Hung, Wei-Chien; Yang, Jessica R; Yankaskas, Christopher L; Wong, Bin Sheng; Wu, Pei-Hsun; Pardo-Pastor, Carlos; Serra, Selma A; Chiang, Meng-Jung; Gu, Zhizhan; Wirtz, Denis; Valverde, Miguel A; Yang, Joy T; Zhang, Jin; Konstantopoulos, Konstantinos

    2016-05-17

    Cells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca(2+) level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca(2+) elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments. PMID:27160899

  11. 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. PMID:23885749

  12. 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

  13. Ca2+ signaling in the myocardium by (redox) regulation of PKA/CaMKII

    PubMed Central

    Johnston, Alex S.; Lehnart, Stephan E.; Burgoyne, Joseph R.

    2015-01-01

    Homeostatic cardiac function is maintained by a complex network of interdependent signaling pathways which become compromised during disease progression. Excitation-contraction-coupling, the translation of an electrical signal to a contractile response is critically dependent on a tightly controlled sequence of events culminating in a rise in intracellular Ca2+ and subsequent contraction of the myocardium. Dysregulation of this Ca2+ handling system as well as increases in the production of reactive oxygen species (ROS) are two major contributing factors to myocardial disease progression. ROS, generated by cellular oxidases and by-products of cellular metabolism, are highly reactive oxygen derivatives that function as key secondary messengers within the heart and contribute to normal homeostatic function. However, excessive production of ROS, as in disease, can directly interact with kinases critical for Ca2+ regulation. This post-translational oxidative modification therefore links changes in the redox status of the myocardium to phospho-regulated pathways essential for its function. This review aims to describe the oxidative regulation of the Ca2+/calmodulin-dependent kinase II (CaMKII) and cAMP-dependent protein kinase A (PKA), and the subsequent impact this has on Ca2+ handling within the myocardium. Elucidating the impact of alterations in intracellular ROS production on Ca2+ dynamics through oxidative modification of key ROS sensing kinases, may provide novel therapeutic targets for preventing myocardial disease progression. PMID:26321952

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

    NASA Astrophysics Data System (ADS)

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

    1993-04-01

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

  15. β-Hydroxybutyric sodium salt inhibition of growth hormone and prolactin secretion via the cAMP/PKA/CREB and AMPK signaling pathways in dairy cow anterior pituitary cells.

    PubMed

    Fu, Shou-Peng; Wang, Wei; Liu, Bing-Run; Yang, Huan-Min; Ji, Hong; Yang, Zhan-Qing; Guo, Bin; Liu, Ju-Xiong; Wang, Jian-Fa

    2015-01-01

    β-hydroxybutyric acid (BHBA) regulates the synthesis and secretion of growth hormone (GH) and prolactin (PRL), but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL gene transcription, and GH and PRL secretion in dairy cow anterior pituitary cells (DCAPCs). The results showed that BHBA decreased intracellular cAMP levels and a subsequent reduction in protein kinase A (PKA) activity. Inhibition of PKA activity reduced cAMP response element-binding protein (CREB) phosphorylation, thereby inhibiting GH and PRL transcription and secretion. The effects of BHBA were attenuated by a specific Gαi inhibitor, pertussis toxin (PTX). In addition, intracellular BHBA uptake mediated by monocarboxylate transporter 1 (MCT1) could trigger AMPK signaling and result in the decrease in GH and PRL mRNA translation in DCAPCs cultured under low-glucose and non-glucose condition when compared with the high-glucose group. This study identifies a biochemical mechanism for the regulatory action of BHBA on GH and PRL gene transcription, translation, and secretion in DCAPCs, which may be one of the factors that regulate pituitary function during the transition period in dairy cows. PMID:25690038

  16. The Octadecaneuropeptide ODN Protects Astrocytes against Hydrogen Peroxide-Induced Apoptosis via a PKA/MAPK-Dependent Mechanism

    PubMed Central

    Hamdi, Yosra; Kaddour, Hadhemi; Vaudry, David; Bahdoudi, Seyma; Douiri, Salma; Leprince, Jérôme; Castel, Helene; Vaudry, Hubert; Tonon, Marie-Christine; Amri, Mohamed; Masmoudi-Kouki, Olfa

    2012-01-01

    Astrocytes synthesize and release endozepines, a family of regulatory peptides, including the octadecaneuropeptide (ODN) an endogenous ligand of both central-type benzodiazepine (CBR) and metabotropic receptors. We have recently shown that ODN exerts a protective effect against hydrogen peroxide (H2O2)-induced oxidative stress in astrocytes. The purpose of the present study was to determine the type of receptor and the transduction pathways involved in the protective effect of ODN in cultured rat astrocytes. We have first observed a protective activity of ODN at very low concentrations that was abrogated by the metabotropic ODN receptor antagonist cyclo1–8[DLeu5]OP, but not by the CBR antagonist flumazenil. We have also found that the metabotropic ODN receptor is positively coupled to adenylyl cyclase in astrocytes and that the glioprotective action of ODN upon H2O2-induced astrocyte death is PKA- and MEK-dependent, but PLC/PKC-independent. Downstream of PKA, ODN induced ERK phosphorylation, which in turn activated the expression of the anti-apoptotic gene Bcl-2 and blocked the stimulation by H2O2 of the pro-apoptotic gene Bax. The effect of ODN on the Bax/Bcl-2 balance contributed to abolish the deleterious action of H2O2 on mitochondrial membrane integrity and caspase-3 activation. Finally, the inhibitory effect of ODN on caspase-3 activity was shown to be PKA and MEK-dependent. In conclusion, the present results demonstrate that the potent glioprotective action of ODN against oxidative stress involves the metabotropic ODN receptor coupled to the PKA/ERK-kinase pathway to inhibit caspase-3 activation. PMID:22927932

  17. 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

  18. 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

  19. Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability.

    PubMed

    Pattabiraman, Diwakar R; Bierie, Brian; Kober, Katharina Isabelle; Thiru, Prathapan; Krall, Jordan A; Zill, Christina; Reinhardt, Ferenc; Tam, Wai Leong; Weinberg, Robert A

    2016-03-01

    The epithelial-to-mesenchymal transition enables carcinoma cells to acquire malignancy-associated traits and the properties of tumor-initiating cells (TICs). TICs have emerged in recent years as important targets for cancer therapy, owing to their ability to drive clinical relapse and enable metastasis. Here, we propose a strategy to eliminate mesenchymal TICs by inducing their conversion to more epithelial counterparts that have lost tumor-initiating ability. We report that increases in intracellular levels of the second messenger, adenosine 3',5'-monophosphate, and the subsequent activation of protein kinase A (PKA) induce a mesenchymal-to-epithelial transition (MET) in mesenchymal human mammary epithelial cells. PKA activation triggers epigenetic reprogramming of TICs by the histone demethylase PHF2, which promotes their differentiation and loss of tumor-initiating ability. This study provides proof-of-principle for inducing an MET as differentiation therapy for TICs and uncovers a role for PKA in enforcing and maintaining the epithelial state. PMID:26941323

  20. Regulation of amygdalar PKA by β-arrestin-2/phosphodiesterase-4 complex is critical for fear conditioning

    PubMed Central

    Li, Yuting; Li, Haohong; Liu, Xing; Bao, Guobin; Tao, Yezheng; Wu, Ziyan; Xia, Peng; Wu, Chunfu; Li, Baoming; Ma, Lan

    2009-01-01

    β-arrestins, key regulators of receptor signaling, are highly expressed in the central nervous system, but their roles in brain physiology are largely unknown. Here we show that β-arrestin-2 is critically involved in the formation of associative fear memory and amygdalar synaptic plasticity. In response to fear conditioning, β-arrestin-2 translocates to amygdalar membrane where it interacts with PDE-4, a cAMP-degrading enzyme, to inhibit PKA activation. Arrb2−/− mice exhibit impaired conditioned fear memory and long-term potentiation at the lateral amygdalar synapses. Moreover, expression of the β-arrestin-2 in the lateral amygdala of Arrb2−/− mice, but not its mutant form that is incapable of binding PDE-4, restores basal PKA activity and rescues conditioned fear memory. Taken together, our data demonstrate that the feedback regulation of amygdalar PKA activation by β-arrestin-2 and PDE-4 complex is critical for the formation of conditioned fear memory. PMID:19955404

  1. 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

  2. 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

  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. PMID:16690998

  4. Prognostic value of FLT3 mutations in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline monochemotherapy

    PubMed Central

    Barragán, Eva; Montesinos, Pau; Camos, Mireia; González, Marcos; Calasanz, Maria J.; Román-Gómez, José; Gómez-Casares, Maria T.; Ayala, Rosa; López, Javier; Fuster, Óscar; Colomer, Dolors; Chillón, Carmen; Larrayoz, María J.; Sánchez-Godoy, Pedro; González-Campos, José; Manso, Félix; Amador, Maria L.; Vellenga, Edo; Lowenberg, Bob; Sanz, Miguel A.

    2011-01-01

    Background Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established. Design and Methods We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005. Results FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis. Conclusions FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens. PMID:21685470

  5. The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons

    PubMed Central

    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

  6. 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

  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. Bile acid structure-activity relationship: evaluation of bile acid lipophilicity using 1-octanol/water partition coefficient and reverse phase HPLC.

    PubMed

    Roda, A; Minutello, A; Angellotti, M A; Fini, A

    1990-08-01

    Two independent methods have been developed and compared to determine the lipophilicity of a representative series of naturally occurring bile acids (BA) in relation to their structure. The BA included cholic acid (CA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), hyodeoxycholic acid (HDCA), ursocholic acid (UCA), hyocholic acid (HCA), as well as their glycine and taurine amidates. Lipophilicity was determined using a 1-octanol/water shake-flask procedure and the experiments were performed at different pH and ionic strengths and at initial BA concentrations below their critical micellar concentrations (CMC) and the water solubility of the protonated form. The experimental data show that both the protonated (HA) and ionized (A-) forms of BA can distribute in 1-octanol, and consequently a partition coefficient for HA (logP' HA) and for A- (logP' A-) must be defined. An equation to predict a weighted apparent distribution coefficient (D) value as a function of pH and pKa has been developed and fits well with the experimental data. Differences between logP for protonated and ionized species for unconjugated BA were in the order of 1 log unit, which increased to 2 for glycine-amidate BA. The partition coefficient of the A- form increased with Na+ concentration and total ionic strength, suggesting an ion-pair mechanism for its partition into 1-octanol. Lipophilicity was also assessed using reverse phase chromatography (C-18-HPLC), and a capacity factor (K') for ionized species was determined. Despite a broad correlation with the logP data, some BA behaved differently. The logP values showed that the order of lipophilicity was DCA greater than CDCA greater than UDCA greater than HDCA greater than HCA greater than CA greater than UCA for both the protonated and ionized unconjugated and glycine-amidate BA, while the K' data showed an inversion for some BA, i.e., DCA greater than CDCA greater than CA greater than HCA greater than UDCA

  9. 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

  10. Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling.

    PubMed

    Thomas, Carissa M; Hong, Teresa; van Pijkeren, Jan Peter; Hemarajata, Peera; Trinh, Dan V; Hu, Weidong; Britton, Robert A; Kalkum, Markus; Versalovic, James

    2012-01-01

    Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H(2) receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases. PMID:22384111

  11. Improved pKa calculations through flexibility based sampling of a water-dominated interaction scheme

    PubMed Central

    Warwicker, Jim

    2004-01-01

    Ionizable groups play critical roles in biological processes. Computation of pKas is complicated by model approximations and multiple conformations. Calculated and experimental pKas are compared for relatively inflexible active-site side chains, to develop an empirical model for hydration entropy changes upon charge burial. The modification is found to be generally small, but large for cysteine, consistent with small molecule ionization data and with partial charge distributions in ionized and neutral forms. The hydration model predicts significant entropic contributions for ionizable residue burial, demonstrated for components in the pyruvate dehydrogenase complex. Conformational relaxation in a pH-titration is estimated with a mean-field assessment of maximal side chain solvent accessibility. All ionizable residues interact within a low protein dielectric finite difference (FD) scheme, and more flexible groups also access water-mediated Debye-Hückel (DH) interactions. The DH method tends to match overall pH-dependent stability, while FD can be more accurate for active-site groups. Tolerance for side chain rotamer packing is varied, defining access to DH interactions, and the best fit with experimental pKas obtained. The new (FD/DH) method provides a fast computational framework for making the distinction between buried and solvent-accessible groups that has been qualitatively apparent from previous work, and pKa calculations are significantly improved for a mixed set of ionizable residues. Its effectiveness is also demonstrated with computation of the pH-dependence of electrostatic energy, recovering favorable contributions to folded state stability and, in relation to structural genomics, with substantial improvement (reduction of false positives) in active-site identification by electrostatic strain. PMID:15388865

  12. 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

  13. Bulgur processes increase nutrition value: possible role in in-vitro protein digestability, phytic acid, trypsin inhibitor activity and mineral bioavailability.

    PubMed

    Ertaş, Nilgün; Türker, Selman

    2014-07-01

    Changes in the chemical constituents and nutritive quality of chickpea bulgur process, were studied in seeds that were soaked at different time (2, 8 and 12 h), different soaking water pH (pH 4, 6 and 8). Soaking in pH 8 soaking water and 12 h soaking time significantly (p < 0.05) reduced the ash content of chickpea bulgur samples. Compared to the raw material, the protein content and in-vitro protein digestibility increased, but starch, crude fiber, fat and energy values decreased and trypsin inhibitor activity was completely eliminated by bulgur process. As the soaking time increased, the phytic acid content also decreased. The highest total phenolic content was determinated with bulgur samples soaked in pH 4 soaking water. The P, Ca, and K values decreased with increasing soaking time. The HCl-extractability of P, Ca, Mg, Fe and K present in chickpea bulgur samples were significantly higher than the raw chickpea seeds. PMID:24966437

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

    PubMed

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

    2014-03-01

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

  15. 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

  16. How closely do the delta(13)C values of Crassulacean Acid metabolism plants reflect the proportion of CO(2) fixed during day and night?

    PubMed

    Winter, Klaus; Holtum, Joseph A M

    2002-08-01

    The extent to which Crassulacean acid metabolism (CAM) plant delta(13)C values provide an index of the proportions of CO(2) fixed during daytime and nighttime was assessed. Shoots of seven CAM species (Aloe vera, Hylocereus monocanthus, Kalanchoe beharensis, Kalanchoe daigremontiana, Kalanchoe pinnata, Vanilla pauciflora, and Xerosicyos danguyi) and two C(3) species (teak [Tectona grandis] and Clusia sp.) were grown in a cuvette, and net CO(2) exchange was monitored for up to 51 d. In species exhibiting net dark CO(2) fixation, between 14% and 73.3% of the carbon gain occurred in the dark. delta(13)C values of tissues formed inside the cuvette ranged between -28.7 per thousand and -11.6 per thousand, and correlated linearly with the percentages of carbon gained in the light and in the dark. The delta(13)C values for new biomass obtained solely during the dark and light were estimated as -8.7 per thousand and -26.9 per thousand, respectively. For each 10% contribution of dark CO(2) fixation integrated over the entire experiment, the delta(13)C content of the tissue was, thus, approximately 1.8 per thousand less negative. Extrapolation of the observations to plants previously surveyed under natural conditions suggests that the most commonly expressed version of CAM in the field, "the typical CAM plant," involves plants that gain about 71% to 77% of their carbon by dark fixation, and that the isotopic signals of plants that obtain one-third or less of their carbon in the dark may be confused with C(3) plants when identified on the basis of carbon isotope content alone. PMID:12177497

  17. Valuing Essays: Essaying Values

    ERIC Educational Resources Information Center

    Badley, Graham

    2010-01-01

    The essay regularly comes under attack. It is criticised for being rigidly linear rather than flexible and reflective. I first challenge this view by examining reasons why the essay should be valued as an important genre. Secondly, I propose that in using the essay form students and academics necessarily exemplify their own critical values. Essays…

  18. Activators of PKA and Epac distinctly influence insulin secretion and cytosolic Ca2+ in female mouse islets stimulated by glucose and tolbutamide.

    PubMed

    Henquin, Jean-Claude; Nenquin, Myriam

    2014-09-01

    Amplification of insulin secretion by cAMP is mediated by protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). Using selective activators, we determined how each effector influences the cytosolic free Ca(2+) concentration ([Ca(2+)]c) and insulin secretion in mouse islets. Alone PKA activator amplified glucose- and tolbutamide-induced insulin secretion, with a greater impact on second than first phase. Epac activator strongly amplified both phases in response to either secretagogue. Amplification was even greater when activators were combined. Although both activators similarly amplified glucose-induced insulin secretion, Epac activator was particularly efficient on tolbutamide-induced insulin secretion. That greater efficacy is attributed to higher [Ca(2+)]c rather than interaction of tolbutamide with Epac, because it was also observed during KCl stimulation. Moreover, in contrast to Epac activator, tolbutamide was inactive when insulin secretion was increased by gliclazide, and its effect on glucose-induced insulin secretion was unaffected by an inhibitor of Epac2. PKA activator increased [Ca(2+)]c during acute or steady-state glucose stimulation, whereas Epac activator had no effect alone or in combination. Neither activator affected [Ca(2+)]c response to tolbutamide or KCl. Metabolic (glucose-mediated) amplification of insulin secretion was unaffected by PKA activator. It was attenuated when insulin secretion was augmented by Epac activator but insensitive to Epac2 inhibitor, which suggests distinct although somewhat overlapping mechanisms. In conclusion, activators of PKA and Epac amplify insulin secretion by augmenting the action of Ca(2+) on exocytosis and, for PKA only, slightly increasing glucose-induced [Ca(2+)]c rise. The influence of Epac seems more important than that of PKA during first phase. PMID:24977470

  19. Dynamic mitochondrial–nuclear redistribution of the immunophilin FKBP51 is regulated by the PKA signaling pathway to control gene expression during adipocyte differentiation

    PubMed Central

    Toneatto, Judith; Guber, Sergio; Charó, Nancy L.; Susperreguy, Sebastián; Schwartz, Jessica; Galigniana, Mario D.; Piwien-Pilipuk, Graciela

    2013-01-01

    Summary Glucocorticoids play an important role in adipogenesis through the glucocorticoid receptor (GR) that forms a heterocomplex with Hsp90•Hsp70 and one high molecular weight immunophilin, either FKBP51 or FKBP52. When 3T3-L1 preadipocytes are induced to differentiate, FKBP51 expression progressively increases, whereas FKBP52 decreases, and Hsp90, Hsp70, p23 and Cyp40 remain unchanged. Interestingly, FKBP51 rapidly translocates from mitochondria to the nucleus where it is retained upon its interaction with chromatin and the nuclear matrix. FKBP51 nuclear localization is transient, and after 48 hours it cycles back to mitochondria. Importantly, this dynamic FKBP51 mitochondrial–nuclear shuttling depends on PKA signaling, because its inhibition by PKI or knockdown of PKA-cα by siRNA, prevented FKBP51 nuclear translocation induced by IBMX. In addition, the electrophoretic pattern of migration of FKBP51 is altered by treatment of cells with PKI or knockdown of PKA-cα, suggesting that FKBP51 is a PKA substrate. In preadipocytes, FKBP51 colocalizes with PKA-cα in mitochondria. When adipogenesis is triggered, PKA-cα also moves to the nucleus colocalizing with FKBP51 mainly in the nuclear lamina. Moreover, FKBP51 and GR interaction increases when preadipocytes are induced to differentiate. GR transcriptional capacity is reduced when cells are incubated in the presence of IBMX, forskolin or dibutyryl-cAMP, compounds that induced FKBP51 nuclear translocation, but not by a specific activator of EPAC. FKBP51 knockdown facilitates adipogenesis, whereas ectopic expression of FKBP51 blocks adipogenesis. These findings indicate that the dynamic mitochondrial–nuclear shuttling of FKBP51 regulated by PKA may be key in fine-tuning the transcriptional control of GR target genes required for the acquisition of adipocyte phenotype. PMID:24101724

  20. Pathway interactions between MAPKs, mTOR, PKA, and the glucocorticoid receptor in lymphoid cells

    PubMed Central

    Miller, Aaron L; Garza, Anna S; Johnson, Betty H; Thompson, E Brad

    2007-01-01

    Background Glucocorticoids are frequently used as a primary chemotherapeutic agent in many types of human lymphoid malignancies because they induce apoptosis through activation of the glucocorticoid receptor, with subsequent alteration of a complex network of cellular mechanisms. Despite clinical usage for over fifty years, the complete mechanism responsible for glucocorticoid-related apoptosis or resistance remains elusive. The mitogen-activated protein kinase pathway is a signal transduction network that influences a variety of cellular responses through phosphorylation of specific target substrates, including the glucocorticoid receptor. In this study we have evaluated the pharmaceutical scenarios which converge on the mitogen-activated protein kinase pathway to alter glucocorticoid sensitivity in clones of human acute lymphoblastic CEM cells sensitive and refractory to apoptosis in response to the synthetic glucocorticoid dexamethasone. Results The glucocorticoid-resistant clone CEM-C1-15 displays a combination of high constitutive JNK activity and dexamethasone-induced ERK activity with a weak induction of p38 upon glucocorticoid treatment. The cells become sensitive to glucocorticoid-evoked apoptosis after: (1) inhibition of JNK and ERK activity, (2) stimulation of the cAMP/PKA pathway with forskolin, or (3) inhibition of mTOR with rapamycin. Treatments 1–3 in combination with dexamethasone alter the intracellular balance of phospho-MAPKs by lowering JNK phosphorylation and increasing the level of glucocorticoid receptor phosphorylated at serine 211, a modification known to enhance receptor activity. Conclusion Our data support the hypothesis that mitogen-activated protein kinases influence the ability of certain malignant lymphoid cells to undergo apoptosis when treated with glucocorticoid. Activated/phosphorylated JNK and ERK appear to counteract corticoid-dependent apoptosis. Inhibiting these MAPKs restores corticoid sensitivity to a resistant clone of

  1. YC-1, a potent antithrombotic agent, induces lipolysis through the PKA pathway in rat visceral fat cells.

    PubMed

    Chin, Chih-Hui; Tsai, Feng-Chou; Chen, Sy-Ping; Wang, Ke-Chuan; Chang, Chao-Chien; Pai, Man-Hui; Fong, Tsorng-Harn

    2012-08-15

    This study investigated the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), a soluble guanylyl cyclase (sGC) activator and potential antithrombotic agent, on lipolysis in isolated visceral fat cells of the rat. Visceral fat cells were isolated from epididymal fat pads of rats and treated with YC-1 at different doses and times. Glycerol release, and intracellular cAMP and cGMP levels were analyzed by specific kits. Moreover, several inhibitors or drugs were used to examine the signal transduction pathways of YC-1-induced lipolysis in adipocytes. Herein we report that YC-1 stimulated glycerol release in dose- and time-dependent manners. Intracellular cAMP and cGMP levels of adipocytes both increased in time-dependent manners, but elevation of the cGMP level was faster and higher than that of the cAMP level after YC-1 treatment. An sGC inhibitor (ODQ) inhibited YC-1-induced glycerol release, indicating the involvement of sGC in YC-1-induced lipolysis. Administration of insulin, an activator of type-3B phosphodiesterase (PDE-3B), attenuated YC-1-induced lipolysis, indicating that elevation of the cAMP level is an important step in the lipolytic effect of YC-1. In addition, YC-1-induced lipolysis was inhibited by a protein kinase A (PKA) inhibitor (KT5720) but not by a PKG inhibitor (KT5823), indicating that YC-1-induced lipolysis occurs through a PKA-dependent pathway. A Western blot analysis showed that extracellular signal-regulated kinase was not phosphorylated by YC-1 treatment. In conclusion, our results suggest that YC-1 might stimulate lipolysis via activation of sGC/cGMP and then activation of the cAMP/PKA signaling cascade in isolated rat visceral adipocytes. PMID:22659114

  2. 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

  3. Phosphorylation by PKA and Cdk5 Mediates the Early Effects of Synapsin III in Neuronal Morphological Maturation.

    PubMed

    Piccini, Alessandra; Perlini, Laura E; Cancedda, Laura; Benfenati, Fabio; Giovedì, Silvia

    2015-09-23

    Synapsin III (SynIII) is a neuron-specific phosphoprotein that plays a unique role in neuronal development. SynIII is phosphorylated by cAMP-dependent protein kinase (PKA) at a highly conserved phosphorylation site and by cyclin-dependent kinase-5 (Cdk5) at a newly described site. Although SynIII is known to be involved in axon elongation in vitro, the role of its phosphorylation by PKA and Cdk5 in the modulation of this process is unknown. We expressed either wild-type (WT) or phosphorylation-site mutants of SynIII in primary SynIII knock-out (KO) mouse neurons at early stages of in vitro development. Whereas the neurite elongation phenotype of SynIII KO neurons was fully rescued by the expression of WT SynIII, the expression of nonphosphorylatable and pseudo-phosphorylated PKA mutants was ineffective. Also, the nonphosphorylatable Cdk5 mutant was unable to rescue the neurite elongation phenotype of SynIII KO neurons. By contrast, the pseudo-phosphorylated mutant rescued the delay in neuronal maturation and axonal elongation, revealing a Cdk5-dependent regulation of SynIII function. Interestingly, SynIII KO neurons also exhibited decreased survival that was fully rescued by the expression of WT SynIII, but not by its phosphorylation mutants, and was associated with increased activated caspase3 and altered tropomyosin receptor kinase B isoform expression. These results indicate that PKA and Cdk5 phosphorylation is required for the physiological action of SynIII on axon specification and neurite outgrowth and that the expression of a functional SynIII is crucial for cell survival. Significance statement: Synapsin III is an atypical member of the synapsin family of synaptic vesicle-associated phosphoproteins that is precociously expressed in neurons and is downregulated afterward. Although experimental evidence suggests a specific role for Synapsin III in neuronal development, the molecular mechanisms are still largely unknown. We found that Synapsin III plays a

  4. Frequency of occurrence of LCS per cascade in bcc iron by PKA energy 30 keV

    NASA Astrophysics Data System (ADS)

    Elias, A.; Driss Khodja, M.

    2009-11-01

    The frequency of occurrence per cascade in bcc iron is studied by PKA energy 30keV using the fitting of potentials on linear collision sequences (LCS). Two embedded atom potentials that essentially differ by their repulsive branches are used in classical molecular dynamic (MD) with the code DYMOKA and its binary collision approximation (BCA) The Molière and Born Mayer potentials are used in the Binary Collision Approximation (BCA). The linear collision sequences (LCS) in iron can be generated with Marlowe code in BCA, using the potentials parameters. The BCA is used to accumulate cascade statistics and to build linear collision sequences distributions.

  5. Aluminium chloride impairs long-term memory and downregulates cAMP-PKA-CREB signalling in rats.

    PubMed

    Zhang, Lifeng; Jin, Cuihong; Lu, Xiaobo; Yang, Jinghua; Wu, Shengwen; Liu, Qiufang; Chen, Rong; Bai, Chunyu; Zhang, Di; Zheng, Linlin; Du, Yanqiu; Cai, Yuan

    2014-09-01

    Epidemiological investigations have indicated that aluminium (Al) is an important environmental neurotoxicant that may be involved in the aetiology of the cognitive dysfunction associated with neurodegenerative diseases. Additionally, exposure to Al is known to cause neurobehavioural abnormalities in animals. Previous studies demonstrated that Al impaired early-phase long-term potentiation (E-LTP) in vivo and in vitro. Our previous research revealed that Al could impair long-term memory via the impairment of late-phase long-term potentiation (L-LTP) in vivo. However, the exact mechanism by which Al impairs long-term memory has been poorly studied thus far. This study was designed not only to observe the effects of subchronic Al treatment on long-term memory and hippocampal ultrastructure but also to explore a possible underlying mechanism (involving the cAMP-PKA-CREB signalling pathway) in the hippocampus of rats.. Pregnant Wistar rats were assigned to four groups. Neonatal rats were exposed to Al by parental lactation for 3 weeks and then fed with distilled water containing 0, 0.2%, 0.4% or 0.6% Al chloride (AlCl3) for 3 postnatal months. The levels of Al in the blood and hippocampus were quantified by atomic absorption spectrophotometry. The shuttle-box test was performed to detect long-term memory. The hippocampus was collected for ultrastructure observation, and the level of cAMP-PKA-CREB signalling was examined. The results showed that the Al concentrations in the blood and hippocampus of Al-treated rats were higher than those of the control rats. Al may impair the long-term memory of rats. Hippocampal cAMP, cPKA, pCREB, BDNF and c-jun expression decreased significantly, and the neuronal and synaptic ultrastructure exhibited pathological changes after Al treatment. These results indicated that Al may induce long-term memory damage in rats by inhibiting cAMP-PKA-CREB signalling and altering the synaptic and neuronal ultrastructure in the hippocampus. PMID

  6. 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

  7. Equal sensitivity of Cav1.2 and Cav1.3 channels to the opposing modulations of PKA and PKG in mouse chromaffin cells.

    PubMed

    Mahapatra, Satyajit; Marcantoni, Andrea; Zuccotti, Annalisa; Carabelli, Valentina; Carbone, Emilio

    2012-10-15

    Mouse chromaffin cells (MCCs) express high densities of L-type Ca2+ channels (LTCCs), which control pacemaking activity and catecholamine secretion proportionally to their density of expression. In vivo phosphorylation of LTCCs by cAMP-PKA and cGMP–PKG, regulate LTCC gating in two opposing ways: the cAMP-PKA pathway potentiates while the cGMP–PKG cascade inhibits LTCCs. Despite this, no attempts have been made to answer three key questions related to the two Cav1 isoforms expressed in MCCs (Cav1.2 and Cav1.3): (i) how much are the two Cav1 channels basally modulated by PKA and PKG?, (ii) to what extent can Cav1.2 and Cav1.3 be further regulated by PKA or PKG activation?, and (iii) are the effects of both kinases cumulative when simultaneously active? Here, by comparing the size of L-type currents of wild-type (WT; Cav1.2+Cav1.3) and Cav1.3−/− KO (Cav1.2) MCCs, we provide new evidence that both PKA and PKG pathways affect Cav1.2 and Cav1.3 to the same extent either under basal conditions or induced stimulation. Inhibition of PKA by H89 (5 μM) reduced the L-type current in WT and KO MCCs by∼60%,while inhibition of PKG by KT 5823 (1 μM) increased by∼40% the same current in both cell types. Given that Cav1.2 and Cav1.3 carry the same quantity of Ca2+ currents, this suggests equal sensitivity of Cav1.2 and Cav1.3 to the two basal modulatory pathways. Maximal stimulation of cAMP–PKA by forskolin (100 μM) and activation of cGMP–PKG by pCPT-cGMP (1mM) uncovered a∼25% increase of L-type currents in the first case and∼65% inhibition in the second case in both WT and KO MCCs, suggesting equal sensitivity of Cav1.2 and Cav1.3 during maximal PKA or PKG stimulation. The effects of PKA and PKG were cumulative and most evident when one pathway was activated and the other was inhibited. The two extreme combinations(PKA activation–PKG inhibition vs. PKG activation-PKA inhibition) varied the size of L-type currents by one order of magnitude (from 180% to 18

  8. Equal sensitivity of Cav1.2 and Cav1.3 channels to the opposing modulations of PKA and PKG in mouse chromaffin cells

    PubMed Central

    Mahapatra, Satyajit; Marcantoni, Andrea; Zuccotti, Annalisa; Carabelli, Valentina; Carbone, Emilio

    2012-01-01

    Mouse chromaffin cells (MCCs) express high densities of L-type Ca2+ channels (LTCCs), which control pacemaking activity and catecholamine secretion proportionally to their density of expression. In vivo phosphorylation of LTCCs by cAMP–PKA and cGMP–PKG, regulate LTCC gating in two opposing ways: the cAMP–PKA pathway potentiates while the cGMP–PKG cascade inhibits LTCCs. Despite this, no attempts have been made to answer three key questions related to the two Cav1 isoforms expressed in MCCs (Cav1.2 and Cav1.3): (i) how much are the two Cav1 channels basally modulated by PKA and PKG?, (ii) to what extent can Cav1.2 and Cav1.3 be further regulated by PKA or PKG activation?, and (iii) are the effects of both kinases cumulative when simultaneously active? Here, by comparing the size of L-type currents of wild-type (WT; Cav1.2 + Cav1.3) and Cav1.3−/− KO (Cav1.2) MCCs, we provide new evidence that both PKA and PKG pathways affect Cav1.2 and Cav1.3 to the same extent either under basal conditions or induced stimulation. Inhibition of PKA by H89 (5 μm) reduced the L-type current in WT and KO MCCs by ∼60%, while inhibition of PKG by KT 5823 (1 μm) increased by ∼40% the same current in both cell types. Given that Cav1.2 and Cav1.3 carry the same quantity of Ca2+ currents, this suggests equal sensitivity of Cav1.2 and Cav1.3 to the two basal modulatory pathways. Maximal stimulation of cAMP–PKA by forskolin (100 μm) and activation of cGMP–PKG by pCPT-cGMP (1 mm) uncovered a ∼25% increase of L-type currents in the first case and ∼65% inhibition in the second case in both WT and KO MCCs, suggesting equal sensitivity of Cav1.2 and Cav1.3 during maximal PKA or PKG stimulation. The effects of PKA and PKG were cumulative and most evident when one pathway was activated and the other was inhibited. The two extreme combinations (PKA activation–PKG inhibition vs. PKG activation–PKA inhibition) varied the size of L-type currents by one order of magnitude

  9. 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

  10. Mechanism of acid reduction at low and high overpotential metal electrodes in the presence and absence of CO2: Implications for CO2 reduction by N-heterocycles

    NASA Astrophysics Data System (ADS)

    Zeitler, Elizabeth L.

    Carbon dioxide reduction is of public interest to synthesize useful materials from CO2 and for storage of renewable energy in a carbon-constrained world. Scientifically, CO2 reduction is of fundamental interest to understand the activation of small molecules and stable chemical bonds. Pyridinium catalysts have been observed to lower the overpotential for reduction of CO2 to methanol at platinum and p-GaP electrodes. In this study, the reduction of pyridinium at a variety of metal electrode surfaces was explored along with its interaction with CO2. The reduction of any weak acid analyte on platinum was found to proceed via a one-electron, proton-coupled process forming H2. The reduction potential could be predicted entirely by acid pKa. Equilibrium and kinetic isotope effects supported this assignment. A prepeak feature observed for acid reductions was examined. Reduction forming a pi-radical was observed for 4,4'-bipyridinium at platinum, gold and glassy carbon via spectroelectrochemistry. Only a small increase in radical decay was observed in the presence of CO 2. Pyridinium reduction at gold was found to occur via proton reduction. Protonated and unprotonated N-heterocycle reductions on glassy carbon can best be explained via pi-reduction. The interaction of CO2 with pyridine was examined. Current in the presence of CO2 was enhanced at slow scan rates due to the slow hydration of CO2 into carbonic acid, leading to pyridinium protonation and is not diagnostic of CO2 reduction. A variety of weak acid analytes showed current enhancement, with greater pKa values leading to greater enhancement. Solution buffering at the electrode interface by CO2 was examined. Current enhancement of pyridinium under CO2 was greater than the sum of the currents for background CO2 reduction and pyridinium reduction, indicating pyridine enhanced CO2 hydration.

  11. Strong-acid, carboxyl-group structures in fulvic acid from the Suwannee River, Georgia. 1. Minor structures

    USGS Publications Warehouse

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

    1995-01-01

    An investigation of the strong-acid characteristics (pKa 3.0 or less) of fulvic acid from the Suwannee River, Georgia, was conducted. Quantitative determinations were made for amino acid and sulfur-containing acid structures, oxalate half-ester structures, malonic acid structures, keto acid structures, and aromatic carboxyl-group structures. These determinations were made by using a variety of spectrometric (13C-nuclear magnetic resonance, infrared, and ultraviolet spectrometry) and titrimetric characterizations on fulvic acid or fulvic acid samples that were chemically derivatized to indicate certain functional groups. Only keto acid and aromatic carboxyl-group structures contributed significantly to the strong-acid characteristics of the fulvic acid; these structures accounted for 43% of the strong-acid acidity. The remaining 57% of the strong acids are aliphatic carboxyl groups in unusual and/or complex configurations for which limited model compound data are available.

  12. Glucose intolerance in dairy goats with pregnancy toxemia: Lack of correlation between blood pH and beta hydroxybutyric acid values.

    PubMed

    Lima, Miguel S; Cota, João B; Vaz, Yolanda M; Ajuda, Inês G; Pascoal, Rita A; Carolino, Nuno; Hjerpe, Charles A

    2016-06-01

    This study assessed the response to a glucose tolerance test in dairy goats with pregnancy toxemia (PT), in healthy, pregnant, non-lactating dairy goats in the last month of gestation (HP), and in healthy, lactating, non-pregnant, dairy goats in mid-lactation (HL). A 500 mL volume of a 5% glucose solution was administered by the IV route. Blood glucose concentrations returned to pre-infusion levels by 90 min in all 8 HL goats, and by 180 min in all 8 HP goats. In contrast, concentrations of blood glucose were still significantly above pre-infusion levels at 180 min post-infusion in all 8 PT goats. Thus, marked glucose intolerance was demonstrated in the PT goats, and mild intolerance was noted in the HP goats. In 25 goats diagnosed with PT and having blood beta hydroxybutyric acid (BHBA) values ≥ 2.9 mmol/L, the correlation coefficient for BHBA with blood pH was non-significant. PMID:27247464

  13. DETERMINATION OF BROMATE IN THE PRESENCE OF BROMINATED HALOACETIC ACIDS BY ION CHROMATOGRAPHY WITH INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRIC DETECTION

    EPA Science Inventory

    Bromate is a disinfection by product (DBP) in drinking water that is formed during the ozonation of a source water containing bromide. Brominated haloacetic acids are DBPs that are anions at near -neutral phs. The anion character of bromoacetic acid (pKa=2.7) is similar to bromat...

  14. Thyroid-Stimulating Hormone Increases HNF-4α Phosphorylation via cAMP/PKA Pathway in the Liver

    PubMed Central

    Song, Yongfeng; Zheng, Dongmei; Zhao, Meng; Qin, Yejun; Wang, Tingting; Xing, Wanjia; Gao, Ling; Zhao, Jiajun

    2015-01-01

    Hepatocyte nuclear factor-4 alpha (HNF-4α) is an orphan nuclear receptor with important roles in hepatic metabolism. Protein phosphorylation plays a functional role in its nuclear localization, DNA binding, and transactivation. Thyroid-stimulating hormone (TSH) is a hormone produced by the anterior pituitary gland, whose direct effect on the metabolic pathway has been observed. Our previous study demonstrated that TSH significantly decreases hepatic nuclear HNF-4α expression. However, whether TSH can influence HNF-4α phosphorylation is unclear. Here, we discovered that TSH can increase HNF-4α phosphorylation and modulate its subcellularlocalization. When HepG2 cells were treated with TSH, the phosphorylation of HNF-4α increased and its nuclear localization was interrupted. Cytoplasmic HNF-4α increased, while nuclear HNF-4α decreased. When the cAMP/PKA pathway was inhibited by the PKA inhibitor H89 and the adenylate cyclase (AC) inhibitor SQ22536, the TSH-mediated phosphorylation of HNF-4α was disrupted. When Tshr was silenced in mice, the phosphorylation of HNF-4α decreased, and cytoplasmic HNF-4α decreased while nuclear HNF-4α increased. In conclusion, our study revealed a novel mechanism by which TSH regulated the hepatic HNF-4α subcellular localization, suggesting the possibility that one of the effects of TSH is to reduce the expression of HNF-4α target genes. PMID:26302721

  15. Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

    PubMed

    Diaz, Miguel F; Li, Nan; Lee, Hyun Jung; Adamo, Luigi; Evans, Siobahn M; Willey, Hannah E; Arora, Natasha; Torisawa, Yu-Suke; Vickers, Dwayne A; Morris, Samantha A; Naveiras, Olaia; Murthy, Shashi K; Ingber, Donald E; Daley, George Q; García-Cardeña, Guillermo; Wenzel, Pamela L

    2015-05-01

    Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5, an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling axis. Blockade of the PGE2-cAMP-PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB). Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. PMID:25870199

  16. PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis

    PubMed Central

    Liu, Shuang; Lai, Li; Zuo, Qiuhong; Dai, Fujun; Wu, Lin; Wang, Yan; Zhou, Qingxia; Liu, Jian; Liu, Jiang; Li, Lei; Lin, Qingxiang; Creighton, Chad J.; Costello, Myra Grace; Huang, Shixia; Jia, Caifeng; Liao, Lujian; Luo, Honglin; Fu, Junjiang; Liu, Mingyao; Yi, Zhengfang; Xiao, Jianru; Li, Xiaotao

    2014-01-01

    The REGγ-proteasome serves as a short-cut for the destruction of certain intact mammalian proteins in the absence of ubiquitin-and ATP. The biological roles of the proteasome activator REGγ are not completely understood. Here we demonstrate that REGγ controls degradation of protein kinase A catalytic subunit-α (PKAca) both in primary human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblast cells (MEFs). Accumulation of PKAca in REGγ-deficient HUVECs or MEFs results in phosphorylation and nuclear exclusion of the transcription factor FoxO1, indicating that REGγ is involved in preserving FoxO1 transcriptional activity. Consequently, VEGF-induced expression of the FoxO1 responsive genes, VCAM-1 and E-Selectin, was tightly controlled by REGγ in a PKA dependent manner. Functionally, REGγ is crucial for the migration of HUVECs. REGγ−/− mice display compromised VEGF-instigated neovascularization in cornea and aortic ring models. Implanted matrigel plugs containing VEGF in REGγ−/− mice induced fewer capillaries than in REGγ+/+ littermates. Taken together, our study identifies REGγ as a novel angiogenic factor that plays an important role in VEGF-induced expression of VCAM-1 and E-Selectin by antagonizing PKA signaling. Identification of the REGγ–PKA–FoxO1 pathway in endothelial cells (ECs) provides another potential target for therapeutic intervention in vascular diseases. PMID:24560667

  17. Correctors of mutant CFTR enhance subcortical cAMP-PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization.

    PubMed

    Abbattiscianni, Anna C; Favia, Maria; Mancini, Maria T; Cardone, Rosa A; Guerra, Lorenzo; Monterisi, Stefania; Castellani, Stefano; Laselva, Onofrio; Di Sole, Francesca; Conese, Massimo; Zaccolo, Manuela; Casavola, Valeria

    2016-03-15

    The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTR-dependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion. PMID:26823603

  18. Biomechanical forces promote blood development through prostaglandin E2 and the cAMP–PKA signaling axis

    PubMed Central

    Diaz, Miguel F.; Li, Nan; Lee, Hyun Jung; Adamo, Luigi; Evans, Siobahn M.; Willey, Hannah E.; Arora, Natasha; Torisawa, Yu-suke; Vickers, Dwayne A.; Morris, Samantha A.; Naveiras, Olaia; Murthy, Shashi K.; Ingber, Donald E.

    2015-01-01

    Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. Here we show that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at embryonic day 9.5, an embryonic stage not previously described to harbor HSCs. Effects on hematopoiesis are mediated in part by a cascade downstream of wall shear stress that involves calcium efflux and stimulation of the prostaglandin E2 (PGE2)–cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA) signaling axis. Blockade of the PGE2–cAMP–PKA pathway in the aorta-gonad-mesonephros (AGM) abolished enhancement in hematopoietic activity. Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element–binding protein (CREB). Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution. These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. PMID:25870199

  19. The anti-diabetic drug repaglinide induces vasorelaxation via activation of PKA and PKG in aortic smooth muscle.

    PubMed

    Kim, Hye Won; Li, Hongliang; Kim, Han Sol; Shin, Sung Eun; Jung, Won-Kyo; Ha, Kwon-Soo; Han, Eun-Taek; Hong, Seok-Ho; Choi, Il-Whan; Firth, Amy L; Bang, Hyoweon; Park, Won Sun

    2016-09-01

    We investigated the vasorelaxant effect of repaglinide and its related signaling pathways using phenylephrine (Phe)-induced pre-contracted aortic rings. Repaglinide induced vasorelaxation in a concentration-dependent manner. The repaglinide-induced vasorelaxation was not affected by removal of the endothelium. In addition, application of a nitric oxide synthase inhibitor (L-NAME) and a small-conductance Ca(2+)-activated K(+) (SKCa) channel inhibitor (apamin) did not alter the vasorelaxant effect of repaglinide on endothelium-intact arteries. Pretreatment with an adenylyl cyclase inhibitor (SQ 22536) or a PKA inhibitor (KT 5720) effectively reduced repaglinide-induced vasorelaxation. Also, pretreatment with a guanylyl cyclase inhibitor (ODQ) or a PKG inhibitor (KT 5823) inhibited repaglinide-induced vasorelaxation. However, pretreatment with a voltage-dependent K(+) (Kv) channel inhibitor (4-AP), ATP-sensitive K(+) (KATP) channel inhibitor (glibenclamide), large-conductance Ca(2+)-activated K(+) (BKCa) channel inhibitor (paxilline), or the inwardly rectifying K(+) (Kir) channel inhibitor (Ba(2+)) did not affect the vasorelaxant effect of repaglinide. Furthermore, pretreatment with a Ca(2+) inhibitor (nifedipine) and a sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor (thapsigargin) did not affect the vasorelaxant effect of repaglinide. The vasorelaxant effect of repaglinide was not affected by elevated glucose (50mM). Based on these results, we conclude that repaglinide induces vasorelaxation via activation of adenylyl cyclase/PKA and guanylyl cyclase/PKG signaling pathways independently of the endothelium, K(+) channels, Ca(2+) channels, and intracellular Ca(2+) ([Ca(2+)]i). PMID:27435474

  20. Improved Long-Term Memory via Enhancing cGMP-PKG Signaling Requires cAMP-PKA Signaling

    PubMed Central

    Bollen, Eva; Puzzo, Daniela; Rutten, Kris; Privitera, Lucia; De Vry, Jochen; Vanmierlo, Tim; Kenis, Gunter; Palmeri, Agostino; D'Hooge, Rudi; Balschun, Detlef; Steinbusch, Harry MW; Blokland, Arjan; Prickaerts, Jos

    2014-01-01

    Memory consolidation is defined by the stabilization of a memory trace after acquisition, and consists of numerous molecular cascades that mediate synaptic plasticity. Commonly, a distinction is made between an early and a late consolidation phase, in which early refers to the first hours in which labile synaptic changes occur, whereas late consolidation relates to stable and long-lasting synaptic changes induced by de novo protein synthesis. How these phases are linked at a molecular level is not yet clear. Here we studied the interaction of the cyclic nucleotide-mediated pathways during the different phases of memory consolidation in rodents. In addition, the same pathways were studied in a model of neuronal plasticity, long-term potentiation (LTP). We demonstrated that cGMP/protein kinase G (PKG) signaling mediates early memory consolidation as well as early-phase LTP, whereas cAMP/protein kinase A (PKA) signaling mediates late consolidation and late-phase-like LTP. In addition, we show for the first time that early-phase cGMP/PKG signaling requires late-phase cAMP/PKA-signaling in both LTP and long-term memory formation. PMID:24813825

  1. What Value "Value Added"?

    ERIC Educational Resources Information Center

    Richards, Andrew

    2015-01-01

    Two quantitative measures of school performance are currently used, the average points score (APS) at Key Stage 2 and value-added (VA), which measures the rate of academic improvement between Key Stage 1 and 2. These figures are used by parents and the Office for Standards in Education to make judgements and comparisons. However, simple…

  2. Setting an Upper Limit on the Myoglobin Iron(IV)Hydroxide pKa: Insight into Axial Ligand Tuning in Heme Protein Catalysis

    PubMed Central

    2015-01-01

    To provide insight into the iron(IV)hydroxide pKa of histidine ligated heme proteins, we have probed the active site of myoglobin compound II over the pH range of 3.9–9.5, using EXAFS, Mössbauer, and resonance Raman spectroscopies. We find no indication of ferryl protonation over this pH range, allowing us to set an upper limit of 2.7 on the iron(IV)hydroxide pKa in myoglobin. Together with the recent determination of an iron(IV)hydroxide pKa ∼ 12 in the thiolate-ligated heme enzyme cytochrome P450, this result provides insight into Nature’s ability to tune catalytic function through its choice of axial ligand. PMID:24875119

  3. mTOR pathway is activated by PKA in adrenocortical cells and participates in vivo to apoptosis resistance in primary pigmented nodular adrenocortical disease (PPNAD).

    PubMed

    de Joussineau, Cyrille; Sahut-Barnola, Isabelle; Tissier, Frédérique; Dumontet, Typhanie; Drelon, Coralie; Batisse-Lignier, Marie; Tauveron, Igor; Pointud, Jean-Christophe; Lefrançois-Martinez, Anne-Marie; Stratakis, Constantine A; Bertherat, Jérôme; Val, Pierre; Martinez, Antoine

    2014-10-15

    Primary pigmented nodular adrenocortical disease (PPNAD) is associated with inactivating mutations of the PRKAR1A tumor suppressor gene that encodes the regulatory subunit R1α of the cAMP-dependent protein kinase (PKA). In human and mouse adrenocortical cells, these mutations lead to increased PKA activity, which results in increased resistance to apoptosis that contributes to the tumorigenic process. We used in vitro and in vivo models to investigate the possibility of a crosstalk between PKA and mammalian target of rapamycin (mTOR) pathways in adrenocortical cells and its possible involvement in apoptosis resistance. Impact of PKA signaling on activation of the mTOR pathway and apoptosis was measured in a mouse model of PPNAD (AdKO mice), in human and mouse adrenocortical cell lines in response to pharmacological inhibitors and in PPNAD tissues by immunohistochemistry. AdKO mice showed increased mTOR complex 1 (mTORC1) pathway activity. Inhibition of mTORC1 by rapamycin restored sensitivity of adrenocortical cells to apoptosis in AdKO but not in wild-type mice. In both cell lines and mouse adrenals, rapid phosphorylation of mTORC1 targets including BAD proapoptotic protein was observed in response to PKA activation. Accordingly, BAD hyperphosphorylation, which inhibits its proapoptotic activity, was increased in both AdKO mouse adrenals and human PPNAD tissues. In conclusion, mTORC1 pathway is activated by PKA signaling in human and mouse adrenocortical cells, leading to increased cell survival, which is correlated with BAD hyperphosphorylation. These alterations could be causative of tumor formation. PMID:24865460

  4. 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. PMID:25846236

  5. Investigations of acidity and nucleophilicity of diphenyldithiophosphinate ligands using theory and gas-phase dissociation reactions

    SciTech Connect

    Christopher M. Leavitt; Garold L. Gresham; Michael T. Benson; Jean-Jaques Gaumet; Dean Peterman; John Klaehn; Megan Moser; Frederic Aubriet; Michael J. Van Stipdonk; Gary S. Groenewold

    2008-04-01

    Diphenyldithiophosphinate (DTP) ligands modified with electron-withdrawing trifluoromethyl (TFM) substitutents are of high interest because they have demonstrated potential for exceptional separation of Am3+ from lanthanide3+ cations. Specifically, the bis(ortho-TFM) (L1-) and (ortho-TFM)(meta-TFM) (L2-) derivatives have shown excellent separation selectivity, while the bis(meta-TFM) (L3)- and unmodified DTP (Lu-) did not. Factors responsible for selective coordination have been investigated using density functional theory (DFT) calculations in concert with competitive dissociation reactions in the gas phase. To evaluate the role of (DTP+H) acidity, density functional calculations were used to predict pKa values, which followed the trend of L3 < L2 < L1 < Lu. The order of the TFM-modified (DTP+H) acids was opposite of what would be expected based on the e--withdrawing effects of the TFM group, suggesting that secondary factors are influencing the pKa and nucleophilicity. The relative nucleophilicities of the DTP anions were evaluated by forming metal-mixed ligand complexes in a trapped ion mass spectrometer, and then fragmenting them using competitive collision induced dissociation. Relative to Na+, the unmodified Lu- anion was the strongest nucleophile. Comparing the TFM derivatives, the bis(ortho-TFM) derivative L1- was found to be the strongest nucleophile, while the bis(meta-TFM) L3- was the weakest, a trend consistent with the pKa calculations. DFT modeling of the Na+ complexes suggested that the elevated cation affinity of the L1- and L2- anions was due to donation of electron density from fluorine atoms to the metal center, which was occurring in rotational conformers where the TFM moiety was proximate to the Na+-dithiophosphinate group. Competitive dissociation experiments were performed with the dithiophosphinate anions complexed with europium nitrate species; ionic dissociation of these complexes always produced the TFM-modified dithiophosphinate anions

  6. Age related reference values for urine creatine and guanidinoacetic acid concentration in children and adolescents by gas chromatography-mass spectrometry.

    PubMed

    Valongo, Carla; Cardoso, Maria Luís; Domingues, Pedro; Almeida, Lígia; Verhoeven, Nanda; Salomons, Gajja; Jakobs, Cornelis; Vilarinho, Laura

    2004-10-01

    A new gas chromatography-mass spectrometry method for routine quantification of urine creatine and guanidinoacetic acid (GAA) has been developed to provide a fast, reliable and inexpensive metabolic screening. Our method uses a two-step derivatization procedure which involves a reaction with hexafluoroacetylacetone followed by a reaction with mono-trimethylsilyltrifluoroacetamide. The standard curves showed linearity over a range of 43-4269 micromol/l for GAA and 38-7325 micromol/l for creatine, which covers the range of GAA and creatine normally found in urine. The lower detection limit is 1.54 micromol/l for GAA and 1.22 micromol/l for creatine, whereas the lower quantification limit is 5.04 micromol/l for GAA and 4.19 micromol/l for creatine. This method was also employed to establish reference values for GAA and creatine in healthy infants, children and adolescents based on the analysis of 169 urine samples. Although no sex differences were observed, normal GAA urinary levels and creatine excretion are distinct in age-related subgroups. We identified a statistically significant age difference in two major groups for GAA (children under 4 years, 18-159 micromol/mmol creatinine; and subjects of 5-16 years, 18-130 micromol/mmol creatinine) whereas three groups were discriminated for creatine (children under 4 years, 0.04-1.51 mmol/mmol creatinine; subjects of 5-11 years, 0.04-1.07 mmol/mmol creatinine; and subjects of 12-16 years, 0.04-0.56 mmol/mmol creatinine). PMID:15369749

  7. Structural Features of PbS Nanocube Monolayers upon Treatment with Mono- and Dicarboxylic Acids and Thiols at a Liquid-Air Interface.

    PubMed

    McPhail, Martin R; Campbell, Gavin P; Bedzyk, Michael J; Weiss, Emily A

    2016-07-01

    This paper describes the ordering of PbS nanocubes (NCs) within free-standing monolayers (suspended on acetonitrile), upon exchanging the native oleate ligands for a series of thiolate and carboxylate ligands at the liquid-air interface. Treatment with either carboxylic acids or thiols effectively decreases the inter-NC separation of nearest-neighbor particles without etching the NC surface. Dicarboxylic acids and dithiols bridge neighboring NCs with an interparticle separation that is consistent with fully extended, bridging ligands. Monocarboxylic acids and monothiols separate NCs by an amount governed by their length, with long-chain ligands showing significant intercalation. (1)H NMR spectroscopy shows carboxylic acids are more effective at replacing the native oleate than are thiols, which we ascribe to the lower pKa values of carboxylic acids. The fast exchange that occurs upon treatment with monocarboxylic acids kinetically traps the clusters of particles in nonclosed packed geometries, so monolayers treated with monocarboxylic acids are, on average, less ordered than those treated with monothiols. Ex situ electron microscopy and grazing incidence small-angle X-ray scattering (GISAXS) analyses of deposited films on Si/SiO2 substrates show that NCs exchanged with nonbridging ligands pack more efficiently at long length scales than do NCs exchanged with bridging ligands, due primarily to the creation of defects within the NC lattice in response to the rigidity of the bridging ligand. PMID:27297625

  8. cAMP/PKA enhances interleukin-1β-induced interleukin-6 synthesis through STAT3 in glial cells.

    PubMed

    Tanabe, Kumiko; Kozawa, Osamu; Iida, Hiroki

    2016-01-01

    We previously reported that interleukin (IL)-1β induces IL-6 synthesis via activation of the IκB/NFκB pathway, p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and signal transducer and activator of transcription (STAT)3, but not p44/p42 MAP kinase in rat glioma cell line, C6 cells and that cAMP enhances the IL-6 synthesis. However, the details behind enhancement of IL-1β-induced IL-6 synthesis by cAMP remain to be elucidated. In the present study, we investigated the exact mechanism of cAMP underlying the amplification of IL-1β-induced IL-6 synthesis in C6 cells. 8-Bromo cAMP significantly enhanced IL-1β-induced STAT3 phosphorylation without affecting phosphorylation of IκB, p38 MAP kinase or SAPK/JNK. In addition, we found that forskolin, a direct activator of adenylyl cyclase, significantly enhanced IL-1β-induced STAT3 phosphorylation. Janus family of tyrosine kinase (JAK) inhibitor I markedly suppressed the amplification by 8-bromo cAMP of IL-1β-induced IL-6 release. IL-1β induced JAK2 phosphorylation, and FLLL32, a specific JAK2 inhibitor, significantly reduced IL-1β-stimulated IL-6 release. 4-Cyano-3-methylisoquinoline, an inhibitor of protein kinase A (PKA), significantly attenuated the enhancing effect of 8-bromo cAMP on IL-1β-induced STAT3 phosphorylation. 8-Bromo cAMP markedly induced JAK2 phosphorylation. PKA siRNA transfection reduced enhancement of IL-1β-induced IL-6 release by 8-bromo cAMP. In conclusion, our results strongly suggest that the adenylyl cyclase/cAMP/PKA pathway upregulates IL-1β-induced IL-6 synthesis through enhancement of the JAK2/STAT3 pathway in C6 glioma cells. PMID:26527061

  9. PKA-independent cAMP stimulation of white adipocyte exocytosis and adipokine secretion: modulations by Ca2+ and ATP.

    PubMed

    Komai, Ali M; Brännmark, Cecilia; Musovic, Saliha; Olofsson, Charlotta S

    2014-12-01

    We examined the effects of cAMP, Ca(2+) and ATP on exocytosis and adipokine release in white adipocytes by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of adipokine secretion. 3T3-L1 adipocyte exocytosis proceeded even in the complete absence of intracellular Ca(2+) ([Ca(2+)]i; buffered with BAPTA) provided cAMP (0.1 mm) was included in the intracellular (pipette-filling) solution. Exocytosis typically plateaued within ∼10 min, probably signifying depletion of a releasable vesicle pool. Inclusion of 3 mm ATP in combination with elevation of [Ca(2+)]i to ≥700 nm augmented the rate of cAMP-evoked exocytosis ∼2-fold and exocytosis proceeded for longer periods (≥20 min) than with cAMP alone. Exocytosis was stimulated to a similar extent upon substitution of cAMP by the Epac (exchange proteins activated by cAMP) agonist 8-Br-2'-O-Me-cAMP (1 mm included in the pipette solution). Inhibition of protein kinase A (PKA) by addition of Rp-cAMPS (0.5 mm) to the cAMP-containing pipette solution was without effect. A combination of the adenylate cyclase activator forskolin (10 μm) and the phosphodiesterase inhibitor IBMX (200 μm; forsk-IBMX) augmented adiponectin secretion measured over 30 min 3-fold and 2-fold in 3T3-L1 and human subcutaneous adipocytes, respectively. This effect was unaltered by pre-loading of cells with the Ca(2+) chelator BAPTA-AM and 2-fold amplified upon inclusion of the Ca(2+) ionophore ionomycin (1 μm) in the extracellular solution. Adiponectin release was also stimulated by the membrane-permeable Epac agonist 8-Br-2'-O-Me-cAMP-AM but unaffected by inclusion of the membrane-permeable PKA inhibitor Rp-8-Br-cAMPS (200 μm). The adipokines leptin, resistin and apelin were present in low amounts in the incubation medium (1-6% of measured adiponectin). Adipsin was secreted in substantial quantities (50% of adiponectin concentration) but release of this adipokine was unaffected by forsk

  10. PKA-mediated responses in females' estrous cycle affect cocaine-induced responses in dopamine-mediated intracellular cascades.

    PubMed

    Weiner, J; Sun, W Lun; Zhou, L; Kreiter, C M; Jenab, S; Quiñones-Jenab, V

    2009-07-01

    An extensive body of literature provides evidence for both sexual dimorphism and menstrual cycle effects in drug abuse patterns and behavioral responses. However, the cellular mechanisms underlying sexually dimorphic responses to and hormonal effects on cocaine use remain unclear. We hypothesized that endogenous hormonal fluctuations during the estrous cycle of rats modulate cocaine's effects on dopamine- and PKA-mediated intracellular responses. To test this hypothesis, intact female rats at different stages of their cycle received a single injection of saline or cocaine (20 mg/kg) and were sacrificed after 15 or 60 min. The nucleus accumbens (NAc) and caudate putamen (CPu) were dissected and analyzed via Western blot for total and phosphorylated (p-thr34) dopamine- and 3'-5'-cyclic AMP-regulated phosphoprotein with molecular weight 32 kDa (DARPP-32), PP1, PP2B (CNA1 and CNB1 subunits), PKA, CREB, cFOS, and Delta-FosB. Our results show that saline-treated rats had estrous cycle-related differences in protein levels of pCREB, DARPP-32, p-thr34-DARPP-32, PP1, and CNA1. Saline-treated female rats in the estrus stage had higher levels of pCREB in the NAc, but cocaine-treatment lowered pCREB levels. The estrous cycle also significantly affected the magnitude of change for p-thr34-DARPP-32 protein levels in both the NAc and CPu. Sixty minutes of cocaine administration increased p-thr34-DARPP-32 levels in the NAc of rats during estrus and proestrus and in the CPu of rats in diestrus. Furthermore, cocaine-induced changes in PP1 protein levels in the NAc were also affected by the stage of the cycle; 60 min of cocaine administration increased PP1 levels in the NAc of rats during diestrus, whereas PP-1 levels decreased in rats during estrus. Taken together, these novel findings suggest that hormonal fluctuations during the estrous cycle may contribute to the previously reported sex differences in the PKA pathway and in behavioral responses to cocaine. PMID:19348873

  11. T-2 toxin regulates steroid hormone secretion of rat ovarian granulosa cells through cAMP-PKA pathway.

    PubMed

    Wu, Jing; Tu, Di; Yuan, Li-Yun; Yi, Jin-e; Tian, Yanan

    2015-02-01

    T-2 toxin is a secondary metabolite produced by Fusarium genus and is a common contaminant in food and feedstuffs of cereal origin. In porcine granulosa cells(GC), T-2 toxin has been shown to inhibit the steroidogenesis; however, the mechanism has not been well understood. Gonadotropin-stimulated steroidogenesis is regulated by the cAMP-PKA pathway. In this study, we investigated potential mechanisms for T-2 toxin-induced reproductive toxicity focusing on the critical steps of the cAMP-PKA pathway affected by T-2 toxin. We first analyzed the effects of T-2 toxin on progesterone and estrogen production in rat granulosa cells. For this purpose the granulosa cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 24h in serum-free medium containing FSH (10 ng/ml) and androstenedione (3 ng/ml), both are required for normal steroidogenesis. Treatment of these cells with T-2 toxin dose-dependently inhibited the growth of cells and the steroid hormone production. Cellular cyclic AMP levels were dose-dependently inhibited by T-2 toxin (0, 1, 10 and 100 nM, 24 h). Furthermore, we found that although the induction of progesterone by 8-Br-cAMP (a FSH mimetic) and 22R-HC (substrate for progesterone) could both be inhibited by T-2 toxin treatment, the T-2-imposed inhibitory effects could be reversed by increasing doses of 22R-HC, while increasing 8-Br-cAMP had no effects, suggesting that T2 toxin targeted at distinct mechanisms. cAMP-stimulated steroidogenic acute regulatory protein (StAR) is a rate limiting protein in progesterone synthesis. Exposure to T2 toxin caused significant suppression of StAR expression as determined by Western blotting and semi-quantitative RT-PCR suggesting StAR is a sensitive target for T-2 toxin. Taken together, our results strongly suggest that T2 toxin inhibits steroidogenesis by suppressing cAMP-PKA pathway and StAR is a target for T-2-toxin. The antisteroidogenesis effects were observable at low T-2 dose (1 ng

  12. PKA-independent cAMP stimulation of white adipocyte exocytosis and adipokine secretion: modulations by Ca2+ and ATP

    PubMed Central

    Komai, Ali M; Brännmark, Cecilia; Musovic, Saliha; Olofsson, Charlotta S

    2014-01-01

    We examined the effects of cAMP, Ca2+ and ATP on exocytosis and adipokine release in white adipocytes by a combination of membrane capacitance patch-clamp recordings and biochemical measurements of adipokine secretion. 3T3-L1 adipocyte exocytosis proceeded even in the complete absence of intracellular Ca2+ ([Ca2+]i; buffered with BAPTA) provided cAMP (0.1 mm) was included in the intracellular (pipette-filling) solution. Exocytosis typically plateaued within ∼10 min, probably signifying depletion of a releasable vesicle pool. Inclusion of 3 mm ATP in combination with elevation of [Ca2+]i to ≥700 nm augmented the rate of cAMP-evoked exocytosis ∼2-fold and exocytosis proceeded for longer periods (≥20 min) than with cAMP alone. Exocytosis was stimulated to a similar extent upon substitution of cAMP by the Epac (exchange proteins activated by cAMP) agonist 8-Br-2′-O-Me-cAMP (1 mm included in the pipette solution). Inhibition of protein kinase A (PKA) by addition of Rp-cAMPS (0.5 mm) to the cAMP-containing pipette solution was without effect. A combination of the adenylate cyclase activator forskolin (10 μm) and the phosphodiesterase inhibitor IBMX (200 μm; forsk–IBMX) augmented adiponectin secretion measured over 30 min 3-fold and 2-fold in 3T3-L1 and human subcutaneous adipocytes, respectively. This effect was unaltered by pre-loading of cells with the Ca2+ chelator BAPTA-AM and 2-fold amplified upon inclusion of the Ca2+ ionophore ionomycin (1 μm) in the extracellular solution. Adiponectin release was also stimulated by the membrane-permeable Epac agonist 8-Br-2′-O-Me-cAMP-AM but unaffected by inclusion of the membrane-permeable PKA inhibitor Rp-8-Br-cAMPS (200 μm). The adipokines leptin, resistin and apelin were present in low amounts in the incubation medium (1–6% of measured adiponectin). Adipsin was secreted in substantial quantities (50% of adiponectin concentration) but release of this adipokine was unaffected by forsk–IBMX. We

  13. Multinuclear nuclear magnetic resonance spectroscopic and high-performance liquid chromatographic characterization of silica, grafted with specifically deuterated 4-((propylamino)methyl)benzoic acid.

    PubMed

    Gangoda, Mahinda E; Wijekoon, Asanka; Gregory, Roger B; Khitrin, Anatoly K

    2016-08-01

    Specifically deuterated 4-((propylamino)methyl)benzoic acid-grafted silica (PAMBA-silica) was prepared by benzylation of propylamino-grafted silica (PA-silica) by either in situ reduction by sodium cyanoborodeuteride (NaCNBD3) of the Schiff base, formed by the reaction between PA-silica and 4-formylbenzoic acid, or by NaCNBD3 reduction of the isolated Schiff base. The PAMBA-silicas, which contain amine and carboxylic acid functionalities, were characterized by elemental analysis, (13)C, (29)Si, and (2)H solid state NMR, and HPLC. Solid state (13)C NMR revealed that PAMBA-silica prepared by the in situ method consists of di-benzylated, mono-benzylated, and unreacted amino-groups while PAMBA-silica prepared by the two-step synthesis consists of only mono-benzylated and unreacted amino-groups. (29)Si solid-state NMR spectra indicated that no significant loss of propylamine groups had occurred during benzylation. Nearly ideal uniaxial rigid-limit (2)H NMR spectra of grafted 4-PAMBA ligands indicates that they form a rigid structure, which provides effective electrostatic screening of inner positive charges when the ligands are in zwitterionic form. HPLC columns packed with PAMBA-silica and PA-silica were evaluated for ionic solutes at different pH of the mobile phase. Retention times increased for cations and decreased for anions at increasing pH. These trends show that PAMBA-silicas act as cation and anion exchangers at high and low pH, respectively. The pKa values of grafted carboxylic acid, determined from HPLC of weakly retaining solutes, are close to pKa of the solution PAMBA. PMID:27372413

  14. Hydroxysafflor yellow A suppress oleic acid-induced acute lung injury via protein kinase A

    SciTech Connect

    Wang, Chaoyun; Huang, Qingxian; Wang, Chunhua; Zhu, Xiaoxi; Duan, Yunfeng; Yuan, Shuai; Bai, Xianyong

    2013-11-01

    Inflammation response and oxidative stress play important roles in acute lung injury (ALI). Activation of the cAMP/protein kinase A (PKA) signaling pathway may attenuate ALI by suppressing immune responses and inhibiting the generation of reactive oxygen species (ROS). Hydroxysafflor yellow A (HSYA) is a natural flavonoid compound that reduces oxidative stress and inflammatory cytokine-mediated damage. In this study, we examined whether HSYA could protect the lungs from oleic acid (OA)-induced injury, which was used to mimic ALI, and determined the role of the cAMP/PKA signaling pathway in this process. Arterial oxygen tension (PaO{sub 2}), carbon dioxide tension, pH, and the PaO{sub 2}/fraction of inspired oxygen ratio in the blood were detected using a blood gas analyzer. We measured wet/dry lung weight ratio and evaluated tissue morphology. The protein and inflammatory cytokine levels in the bronchoalveolar lavage fluid and serum were determined using enzyme-linked immunoassay. The activities of superoxide dismutase, glutathione peroxidase, PKA, and nicotinamide adenine dinucleotide phosphate oxidase, and the concentrations of cAMP and malondialdehyde in the lung tissue were detected using assay kits. Bcl-2, Bax, caspase 3, and p22{sup phox} levels in the lung tissue were analyzed using Western blotting. OA increased the inflammatory cytokine and ROS levels and caused lung dysfunction by decreasing cAMP synthesis, inhibiting PKA activity, stimulating caspase 3, and reducing the Bcl-2/Bax ratio. H-89 increased these effects. HSYA significantly increased the activities of antioxidant enzymes, inhibited the inflammatory response via cAMP/PKA pathway activation, and attenuated OA-induced lung injury. Our results show that the cAMP/PKA signaling pathway is required for the protective effect of HSYA against ALI. - Highlights: • Oleic acid (OA) cause acute lung injury (ALI) via inhibiting cAMP/PKA signal pathway. • Blocking protein kinase A (PKA) activation may

  15. Connectivity patterns and rotamer states of nucleobases determine acid-base properties of metalated purine quartets.

    PubMed

    Lüth, Marc Sven; Freisinger, Eva; Kampf, Gunnar; Garijo Anorbe, Marta; Griesser, Rolf; Operschall, Bert P; Sigel, Helmut; Lippert, Bernhard

    2015-07-01

    Potentiometric pH titrations and pD dependent (1)H NMR spectroscopy have been applied to study the acidification of the exocyclic amino group of adenine (A) model nucleobases (N9 position blocked by alkyl groups) when carrying trans-a2Pt(II) (with a=NH3 or CH3NH2) entities both at N1 and N7 positions. As demonstrated, in trinuclear complexes containing central A-Pt-A units, it depends on the connectivity pattern of the adenine bases (N7/N7 or N1/N1) and their rotamer states (head-head or head-tail), how large the acidifying effect is. Specifically, a series of trinuclear complexes with (A-N7)-Pt-(N7-A) and (A-N1)-Pt-(N1-A) cross-linking patterns and terminal 9-alkylguanine ligands (9MeGH, 9EtGH) have been analyzed in this respect, and it is shown that, for example, the 9MeA ligands in trans-,trans-,trans-[Pt(NH3)2(N7-9MeA-N1)2{Pt(NH3)2(9EtGH-N7)}2](ClO4)6·6H2O (4a) and trans-,trans-,trans-[Pt(NH3)2(N7-9EtA-N1)2{Pt(CH3NH2)2(9-MeGH-N7)}2](ClO4)6·3H2O (4b) are more acidic, by ca. 1.3 units (first pKa), than the linkage isomer trans-,trans-,trans-[Pt(CH3NH2)2(N1-9MeA-N7)2{Pt(NH3)2(9MeGH-N7)}2](NO3)6·6.25H2O (1b). Overall, acidifications in these types of complexes amount to 7-9 units, bringing the pKa values of such adenine ligands in the best case close to the physiological pH range. Comparison with pKa values of related trinuclear Pt(II) complexes having different co-ligands at the Pt ions, confirms this picture and supports our earlier proposal that the close proximity of the exocyclic amino groups in a head-head arrangement of (A-N7)-Pt-(N7-A), and the stabilization of the resulting N6H(-)⋯H2N6 unit, is key to this difference. PMID:25773716

  16. Toxicity of chlorinated phenoxyacetic acid herbicides in the experimental eukaryotic model Saccharomyces cerevisiae: role of pH and of growth phase and size of the yeast cell population.

    PubMed

    Cabral, M G; Viegas, C A; Teixeira, M C; Sá-Correia, I

    2003-04-01

    The inhibitory effect of the herbicides 2-methyl-4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in Saccharomyces cerevisiae growth is strongly dependent on medium pH (range 2.5-6.5). Consistent with the concept that the toxic form is the liposoluble undissociated form, at values close to their pK(a) (3.07 and 2.73, respectively) the toxicity is high, decreasing with the increase of external pH. In addition, the toxicity of identical concentrations of the undissociated acid form is pH independent, as observed with 2,4-dichlorophenol (2,4-DCP), an intermediate of 2,4-D degradation. Consequently, at pH values above 3.5 (approximately one unit higher than 2,4-D pK(a)), 2,4-DCP becomes more toxic than the original herbicide. A dose-dependent inhibition of growth kinetics and increased duration of growth latency is observed following sudden exposure of an unadapted yeast cell population to the presence of the herbicides. This contrasts with the effect of 2,4-DCP, which essentially affects growth kinetics. Experimental evidences suggest that the acid herbicides toxicity is not exclusively dependent on the liposolubility of the toxic form, as may essentially be the case of 2,4-DCP. An unadapted yeast cell population at the early stationary-phase of growth under nutrient limitation is significantly more resistant to short-term herbicide induced death than an exponential-phase population. Consequently, the duration of growth latency is reduced, as observed with the increase of the size of the herbicide stressed population. However, these physiological parameters have no significant effect either on growth kinetics, following growth resumption under herbicide stress, or on the growth curve of yeast cells previously adapted to the herbicides, indicating that their role is exerted at the level of cell adaptation. PMID:12586155

  17. Hydrogelation and Crystallization of Sodium Deoxycholate Controlled by Organic Acids.

    PubMed

    Li, Guihua; Hu, Yuanyuan; Sui, Jianfei; Song, Aixin; Hao, Jingcheng

    2016-02-16

    The gelation and crystallization behavior of a biological surfactant, sodium deoxycholate (NaDC), mixed with l-taric acid (L-TA) in water is described in detail. With the variation of molar ratio of L-TA to NaDC (r = nL-TA/nNaDC) and total concentration of the mixtures, the transition from sol to gel was observed. SEM images showed that the density of nanofibers gradually increases over the sol-gel transition. The microstructures of the hydrogels are three-dimensional networks of densely packed nanofibers with lengths extending to several micrometers. One week after preparation, regular crystallized nanospheres formed along the length of the nanofibers, and it was typical among the transparent hydrogels induced by organic acids with pKa1 value <3.4. Small-angle X-ray diffraction demonstrated differences in the molecular packing between transparent and turbid gels, indicating a variable hydrogen bond mode between NaDC molecules. PMID:26783993

  18. PKA RIα Homodimer Structure Reveals an Intermolecular Interface with Implications for Cooperative cAMP Binding and Carney Complex Disease

    PubMed Central

    Bruystens, Jessica G.H.; Wu, Jian; Fortezzo, Audrey; Kornev, Alexandr P.; Blumenthal, Donald K.; Taylor, Susan S.

    2014-01-01

    Summary The regulatory (R) subunit is the cAMP receptor of protein kinase A. Following cAMP binding, the inactive PKA holoenzyme complex separates into two active catalytic (C) subunits and a cAMP-bound R dimer. Thus far, only monomeric R structures have been solved, which fell short in explaining differences of cAMP binding for the full-length protein as compared to the truncated R subunits. Here we solved a full-length R-dimer structure that reflects the biologically relevant conformation, and this structure agrees well with small angle X-ray scattering. An isoform-specific interface is revealed between the protomers. This interface acts as an intermolecular sensor for cAMP and explains the cooperative character of cAMP binding to the RIα dimer. Mutagenesis of residues on this interface not only leads to structural and biochemical changes, but is also linked to Carney complex disease. PMID:24316401

  19. Reversal of the surface charge asymmetry in purple membrane due to single amino acid substitutions.

    PubMed Central

    Hsu, K C; Rayfield, G W; Needleman, R

    1996-01-01

    Twenty-seven mutant bacteriorhodopsin's were screened to determine the PKa for reversal of the permanent electric dipole moment. The photoelectric response of an aqueous purple-membrane suspension was used to determine the direction of the purple-membrane dipole moment as a function of pH. The pK(a) for the dipole reversal of wild-type bacteriorhodopsin is 4.5. Six of the 27 mutant bacteriorhodopsin's were found to have a pK(a) for dipole reversal larger than that of wild-type bacteriorhodopsin. Two of these mutants, L93T and L93W, involve a neutral amino acid substitution in the interior of the protein. The direction of the purple-membrane permanent electric dipole moment is determined by the purple-membrane surface charge asymmetry. We conclude that these two substitutions, which do not involve charge replacement, alter the pK(a) for the reversal of the purple-membrane surface charge asymmetry. We suggest that these changes to the pK(a) are due to altered protein folding at the surface of the purple-membrane induced by single-site substitutions in the protein interior. PMID:9172760

  20. Mouse cystic fibrosis transmembrane conductance regulator forms cAMP-PKA-regulated apical chloride channels in cortical collecting duct.

    PubMed

    Lu, Ming; Dong, Ke; Egan, Marie E; Giebisch, Gerhard H; Boulpaep, Emile L; Hebert, Steven C

    2010-03-30

    The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK. PMID:20231442

  1. Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: Relationship with cell proliferation

    SciTech Connect

    Mantovani, G.; Lania, A.G.; Bondioni, S.; Peverelli, E.; Pedroni, C.; Ferrero, S.; Pellegrini, C.; Vicentini, L.; Arnaldi, G.; Bosari, S.; Beck-Peccoz, P.; Spada, A.

    2008-01-01

    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 growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n = 16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n = 5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

  2. Corticotropin-Releasing Factor Facilitates Epileptiform Activity in the Entorhinal Cortex: Roles of CRF2 Receptors and PKA Pathway

    PubMed Central

    Kurada, Lalitha; Yang, Chuanxiu; Lei, Saobo

    2014-01-01

    Whereas corticotropin-releasing factor (CRF) has been considered as the most potent epileptogenic neuropeptide in the brain, its action site and underlying mechanisms in epilepsy have not been determined. Here, we found that the entorhinal cortex (EC) expresses high level of CRF and CRF2 receptors without expression of CRF1 receptors. Bath application of CRF concentration-dependently increased the frequency of picrotoxin (PTX)-induced epileptiform activity recorded from layer III of the EC in entorhinal slices although CRF alone did not elicit epileptiform activity. CRF facilitated the induction of epileptiform activity in the presence of subthreshold concentration of PTX which normally would not elicit epileptiform activity. Bath application of the inhibitor for CRF-binding proteins, CRF6-33, also increased the frequency of PTX-induced epileptiform activity suggesting that endogenously released CRF is involved in epileptogenesis. CRF-induced facilitation of epileptiform activity was mediated via CRF2 receptors because pharmacological antagonism and knockout of CRF2 receptors blocked the facilitatory effects of CRF on epileptiform activity. Application of the adenylyl cyclase (AC) inhibitors blocked CRF-induced facilitation of epileptiform activity and elevation of intracellular cyclic AMP (cAMP) level by application of the AC activators or phosphodiesterase inhibitor increased the frequency of PTX-induced epileptiform activity, demonstrating that CRF-induced increases in epileptiform activity are mediated by an increase in intracellular cAMP. However, application of selective protein kinase A (PKA) inhibitors reduced, not completely blocked CRF-induced enhancement of epileptiform activity suggesting that PKA is only partially required. Our results provide a novel cellular and molecular mechanism whereby CRF modulates epilepsy. PMID:24505399

  3. Transcriptional regulation of cyclin D2 by the PKA pathway and inducible cAMP early repressor in granulosa cells.

    PubMed

    Muñiz, Luis C; Yehia, Ghassan; Mémin, Elisabeth; Ratnakar, Pillarisetty V A L; Molina, Carlos A

    2006-08-01

    Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis. PMID:16625003

  4. Impaired muscarinic type 3 (M3) receptor/PKC and PKA pathways in islets from MSG-obese rats.

    PubMed

    Ribeiro, Rosane Aparecida; Balbo, Sandra Lucinei; Roma, Letícia Prates; Camargo, Rafael Ludemann; Barella, Luiz Felipe; Vanzela, Emerielle Cristine; de Freitas Mathias, Paulo Cesar; Carneiro, Everardo Magalhães; Boschero, Antonio Carlos; Bonfleur, Maria Lúcia

    2013-07-01

    Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG β-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K(+) (P < 0.05). The MSG group also released less insulin in response to 100 μM carbachol, 10 μM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of β-cell dysfunction. PMID:23652999

  5. GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling

    PubMed Central

    Marroquin-Guzman, Margarita; Wilson, Richard A.

    2015-01-01

    Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development. PMID:25901357

  6. Neuropeptide Y inhibits ciliary beat frequency in human ciliated cells via nPKC, independently of PKA.

    PubMed

    Wong, L B; Park, C L; Yeates, D B

    1998-08-01

    The intracellular mechanisms whereby the inhibitory neurotransmitter neuropeptide Y (NPY) decreases ciliary beat frequency (CBF) were investigated in cultured human tracheal and bronchial ciliated cells. CBF was measured by nonstationary analysis laser light scattering. NPY at 1 and 10 microM decreased CBF from a baseline of 6.7 +/- 0.5 (n = 12) to 6.1 +/- 0.5 (P < 0.05) and 5.8 +/- 0.4 (P < 0.01) Hz, respectively. Prior application of PYX-1, an NPY antagonist, prevented the decreases of CBF induced by both doses of NPY. Two broad protein kinase C (PKC) kinase inhibitors, staurosporine and calphostin C, also abolished the NPY-induced decrease in CBF. The NPY-induced decrease in CBF was abolished by GF 109203X, a novel PKC (nPKC) isoform inhibitor, whereas this decrease in CBF was not attenuated by Gö-6976, a specific inhibitor of conventional PKC isoforms. Because pretreatment with NPY did not block the stimulation of CBF by forskolin and pretreatment with forskolin did not abolish the NPY-induced inhibition of CBF, this NPY receptor-mediated signal transduction mechanism appears to be independent of the adenylate cyclase-protein kinase A (PKA) pathway. Inhibition of Ca2+-ATPase by thapsigargin also prevented the suppression of CBF induced by subsequent application of NPY. These novel data indicate that, in cultured human epithelia, NPY decreases CBF below its basal level via the activation of an nPKC isoform and Ca2+-ATPase, independent of the activity of PKA. This is consistent with the proposition that NPY is an autonomic efferent inhibitory neurotransmitter regulating mucociliary transport. PMID:9688598

  7. Comparison of the influence of polyaspartic acid and polylysine functional groups on the adsorption at the Cr2O3-Aqueous polymer solution interface

    NASA Astrophysics Data System (ADS)

    Ostolska, Iwona; Wiśniewska, Małgorzata

    2014-08-01

    Polyamino acids are a group of synthesized polymers obtained by polymerization of a given kind of amino acid monomer. Because of high biodegradability of this class of polymers, they can be used as flocculation or stabilization agents in the environmental aspects. Therefore determination of their influence on the stability of the aqueous suspension of metal oxides is important. An influence of different functional groups of polyamino acids, their molecular weight and concentration on the adsorption at the chromium (III) oxide (Cr2O3)-aqueous solution interface was determined. Experiments were carried out for four values of solution pH varying from 3 to 10 (3, 4, 7.6 and 10, respectively). Two polymers were used: anionic polyaspartic acid (ASP) of 6800 and 27,000 as well as polylysine (LYS) of 4900 and 33,000 molecular weights. Changes of surface charge density of colloidal Cr2O3 in the presence and in the absence of macromolecular substances were determined using potentiometric titration. In these studies the influence of the concentration and molecular weight of the ionic polymers on the pHpzc value was determined. Additionally, due to the lack of appropriate literature data, potentiometric titration of the selected polymers was performed to determine pKa values.

  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 cyclic AMP (cAMP)-PKA pathway is a central signaling cascade that transmits extracellular stimuli and governs cell responses through the second messenger cAMP. The importance of cAMP signaling in fungal biology has been well documented. Two key conserved components, adenylate cyclase (AC) and ca...

  9. STC1 induction by PACAP is mediated through cAMP and ERK1/2 but not PKA in cultured cortical neurons

    PubMed Central

    Holighaus, Yvonne; Weihe, Eberhard; Eiden, Lee E.

    2011-01-01

    The neuroprotective actions of PACAP (pituitary adenylate cyclase-activating polypeptide) in vitro and in vivo suggest that activation of its cognate G protein-coupled receptor PAC1 or downstream signaling molecules, and thus activation of PACAP target genes, could be of therapeutic benefit. Here we show, that cultured rat cortical neurons predominantly expressed the PAC1hop and null variants, activation of which resulted in elevation of the two second messengers cAMP and Ca2+ and expression of the putative neuroprotectant stanniocalcin 1 (STC1). PACAP signaling to the STC1 gene proceeded through the extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not through the cAMP dependent protein kinase (PKA), and was mimicked by the adenylate cyclase activator forskolin. PACAP- and forskolin-mediated activation of ERK1/2 occurred through cAMP, but not PKA. These results suggest that STC1 gene induction proceeds through cAMP and ERK1/2, independently of PKA, the canonical cAMP effector. In contrast, PACAP signaling to the BDNF gene proceeded through PKA, suggesting that two different neuroprotective cAMP pathways co-exist in differentiated cortical neurons. The selective activation of a potentially neuroprotective cAMP dependent pathway different from the canonical cAMP pathway used in many physiological processes, such as memory storage, has implications for pharmacological activation of neuroprotection in vivo. PMID:21975601

  10. PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

    PubMed Central

    Terrin, Anna; Monterisi, Stefania; Stangherlin, Alessandra; Zoccarato, Anna; Koschinski, Andreas; Surdo, Nicoletta C.; Mongillo, Marco; Sawa, Akira; Jordanides, Niove E.; Mountford, Joanne C.

    2012-01-01

    Previous work has shown that the protein kinase A (PKA)–regulated phosphodiesterase (PDE) 4D3 binds to A kinase–anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA–PDE4D3–AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals. PMID:22908311

  11. Observation of pH Value in Electrokinetic Remediation using various electrolyte (MgSO4, KH2PO4 and Na(NO3)) for Barren Acidic Soil at Ayer Hitam, Johor, Malaysia

    NASA Astrophysics Data System (ADS)

    Norashira, J.; Zaidi, E.; Aziman, M.; Saiful Azhar, A. T.

    2016-07-01

    Barren acidic soil collected at Ayer Hitam, Johor Malaysia was recorded at pH value of 2.36 with relative humidity of 86%. This pH value is not suitable for the growth of any plants especially for the soil stabilization purposes. Gradation weathering within the range of 4 to 6 indicates an incomplete/partial weathering process. The soil grade in this range is known as a black shale mudstone. Beside, this also influences to a factor of the high surface water runoff at this particular soil species. As the acidic pH become a major problem for soil fertilizing hence an appropriate technique was implemented known as using ‘Electrokinetic Remediation’, EKR. This technique has a great potential in changing the soil pH value from acidic to less acidic and also kept maintain the pH at the saturated rate of electrochemical process. This research study presents the monitoring data of pH value due to the effect of various electrolyte consist of 0.5M of MgSO4, KH2PO4, and Na(NO3). Here, the distilled water (DW) was used as reference solution. The electric field was provided by dipping two pieces of identical rectangular aluminum foil as anode and cathode. The EKR was conducted under a constant voltage gradient of 50 V/m across the sample bulk at 0.14 m length measured between both electrodes. The data collection was conducted during the total period of 7 days surveillance. The variation of pH values at the remediation area between anode and cathode for various type of electrolyte indicates that there are a significant saturated value as it reaches 7 days of treatment. During the analysis, it is found that the highest pH value at the remediation area after 7 days treatment using Na(NO3), KH2PO4 and MgSO4 was 3.93, 3.33 and 3.39 respectively. Hence from the last stage of pH value observation, it can be conclude that the best electrolyte for barren soil treatment is Na(NO3) whereby it contribute to highest pH value and turn the soil to be less acidic.

  12. Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo.

    PubMed

    Efetova, Marina; Petereit, Linda; Rosiewicz, Kamil; Overend, Gayle; Haußig, Florian; Hovemann, Bernhard T; Cabrero, Pablo; Dow, Julian A T; Schwärzel, Martin

    2013-02-01

    Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a variety of essential processes in diverse cell types, functioning via cAMP-dependent effectors such as protein kinase A (PKA) and/or exchange proteins directly activated by cAMP (EPAC). In an intact tissue it is difficult to separate the contribution of each cAMP effector in a particular cell type using genetic or pharmacological approaches alone. We, therefore, utilized optogenetics to overcome the difficulties associated with examining a multicellular tissue. The transgenic photoactive adenylyl cyclase bPAC can be activated to rapidly and reversibly generate cAMP pulses in a cell-type-specific manner. This optogenetic approach to cAMP manipulation was validated in vivo using GAL4-driven UAS-bPAC in a simple epithelium, the Drosophila renal (Malpighian) tubules. As bPAC was expressed under the control of cell-type-specific promoters, each cAMP signal could be directed to either the stellate or principal cells, the two major cell types of the Drosophila renal tubule. By combining the bPAC transgene with genetic and pharmacological manipulation of either PKA or EPAC it was possible to investigate the functional impact of PKA and EPAC independently of each other. The results of this investigation suggest that both PKA and EPAC are involved in cAMP sensing, but are engaged in very different downstream physiological functions in each cell type: PKA is necessary for basal secretion in principal cells only, and for stimulated fluid secretion in stellate cells only. By contrast, EPAC is important in stimulated fluid secretion in both cell types. We propose that such optogenetic control of cellular cAMP levels can be applied to other systems, for example the heart or the central nervous system, to investigate the physiological impact of cAMP-dependent signaling pathways with unprecedented precision. PMID:23264735

  13. Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling

    PubMed Central

    Corcoran, K A; Leaderbrand, K; Jovasevic, V; Guedea, A L; Kassam, F; Radulovic, J

    2015-01-01

    In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B–RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions. PMID:26460481

  14. pH-Responsive Behavior of Poly(acrylic acid) Brushes of Varying Thickness

    NASA Astrophysics Data System (ADS)

    Yadav, Vivek; Robertson, Megan; Conrad, Jacinta

    2015-03-01

    We have investigated the pH-dependent response of polyelectrolyte brushes of varying thickness. Our model system consists of poly(acrylic acid) brushes, which change from hydrophobic and neutral at low pH to hydrophilic and negatively charged at high pH, synthesized using a grafting-from approach at constant grafting density. As the polymer brush thickness increased, the brushes exhibited greater hysteresis in static water contact angle as a function of pH. We extracted the pKa of the polymer brushes from contact angle measurements. The relationship between the pKa and brush thickness depended on the order in which the brushes were exposed to solutions of varying pH: pKa decreased on increasing brush thickness when going from basic to acidic medium whereas pKa increased on increasing brush thickness when going from acidic to basic medium. We speculate that the origin of hysteresis can be explained by pH-dependent conformational changes in these polyelectrolyte brushes.

  15. MCCE analysis of the pKas of introduced buried acids and bases in staphylococcal nuclease.

    PubMed

    Gunner, M R; Zhu, Xuyu; Klein, Max C

    2011-12-01

    The pK(a)s of 96 acids and bases introduced into buried sites in the staphylococcal nuclease protein (SNase) were calculated using the multiconformation continuum electrostatics (MCCE) program and the results compared with experimental values. The pK(a)s are obtained by Monte Carlo sampling of coupled side chain protonation and position as a function of pH. The dependence of the results on the protein dielectric constant (ε(prot)) in the continuum electrostatics analysis and on the Lennard-Jones non-electrostatics parameters was evaluated. The pK(a)s of the introduced residues have a clear dependence on ε(prot,) whereas native ionizable residues do not. The native residues have electrostatic interactions with other residues in the protein favoring ionization, which are larger than the desolvation penalty favoring the neutral state. Increasing ε(prot) scales both terms, which for these residues leads to small changes in pK(a). The introduced residues have a larger desolvation penalty and negligible interactions with residues in the protein. For these residues, changing ε(prot) has a large influence on the calculated pK(a). An ε(prot) of 8-10 and a Lennard-Jones scaling of 0.25 is best here. The X-ray crystal structures of the mutated proteins are found to provide somewhat better results than calculations carried out on mutations made in silico. Initial relaxation of the in silico mutations by Gromacs and extensive side chain rotamer sampling within MCCE can significantly improve the match with experiment. PMID:21910138

  16. Some features of the effect the pH value and the physicochemical properties of boric acid have on mass transfer in a VVER reactor's core

    NASA Astrophysics Data System (ADS)

    Gavrilov, A. V.; Kritskii, V. G.; Rodionov, Yu. A.; Berezina, I. G.

    2013-07-01

    Certain features of the effect of boric acid in the reactor coolant of nuclear power installations equipped with a VVER-440 reactor on mass transfer in the reactor core are considered. It is determined that formation of boric acid polyborate complexes begins under field conditions at a temperature of 300°C when the boric acid concentration is equal to around 0.065 mol/L (4 g/L). Operations for decontaminating the reactor coolant system entail a growth of corrosion product concentration in the coolant, which gives rise to formation of iron borates in the zones where subcooled boiling of coolant takes place and to the effect of axial offset anomalies. A model for simulating variation of pressure drop in a VVER-440 reactor's core that has invariable parameters during the entire fuel campaign is developed by additionally taking into account the concentrations of boric acid polyborate complexes and the quantity of corrosion products (Fe, Ni) represented by the ratio of their solubilities.

  17. Low abdominal NIRS values and elevated plasma intestinal fatty acid-binding protein in a premature piglet model of necrotizing enterocolitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To identify early markers of necrotizing enterocolitis (NEC), we hypothesized that continuous abdominal near-infrared spectroscopy (A-NIRS) measurement of splanchnic tissue oxygen saturation and intermittent plasma intestinal fatty-acid binding protein (pI-FABP) measured every 6 hours can detect NEC...

  18. New 4-hydroxy-N-(2-hydroxyethyl)butanamides: Structure and acidity

    NASA Astrophysics Data System (ADS)

    Duarte-Hernández, Angélica M.; Contreras, Rosalinda; Suárez-Moreno, Galdina V.; González, Felipe J.; Flores-Parra, Angelina

    2015-02-01

    Optically active new 4-hydroxy-N-(2-hydroxyethyl)butanamides are reported. The structure in the solid state of four racemic mixtures and three enantiomeric pure compounds was determined by X-ray diffraction. The solid state arrangements of enantiomerically pure compounds were compared with those of their racemic mixtures. The butanamides prefer lineal conformations in the solid state. They form dimers and polymers through intermolecular hydrogen bonds. Variable temperature 1H NMR experiments were performed in order to analyse the strength of the hydrogen bonds in the neutral molecules in solution. The pKa values of the dihydroxybutanamides in DMSO determined by cyclic voltammetric (11.1-14.5) were lower than those reported for amides RNHCOR without hydroxyl groups (25.9). The latter motivated us to investigate the role of hydrogen bonds in the stabilization of the corresponding anions and in consequence in the acidity of the amides. The explanation of the enhanced Nsbnd H acidity of dihydroxybutanamides was found in the cyclic voltammetric experiments, in conductimetric titrations using nBu4NOH in methanol and titration with CsOH in acetonitrile. The optimised structures for the neutral molecules and their mono and dianions calculated by ab-initio HF(6-31 + G∗) and B3LYP(6-31 + G∗) methods supported the explanation of the enhanced Nsbnd H acidity.

  19. Effects of Cardiac Troponin I Mutation P83S on Contractile Properties and the Modulation by PKA-Mediated Phosphorylation.

    PubMed

    Cheng, Yuanhua; Lindert, Steffen; Oxenford, Lucas; Tu, An-Yue; McCulloch, Andrew D; Regnier, Michael

    2016-08-25

    cTnI(P82S) (cTnI(P83S) in rodents) resides at the I-T arm of cardiac troponin I (cTnI) and was initially identified as a disease-causing mutation of hypertrophic cardiomyopathy (HCM). However, later studies suggested this may not be true. We recently reported that introduction of an HCM-associated mutation in either inhibitory-peptide (cTnI(R146G)) or cardiac-specific N-terminus (cTnI(R21C)) of cTnI blunts the PKA-mediated modulation on myofibril activation/relaxation kinetics by prohibiting formation of intrasubunit contacts between these regions. Here, we tested whether this also occurs for cTnI(P83S). cTnI(P83S) increased both Ca(2+) binding affinity to cTn (KCa) and affinity of cTnC for cTnI (KC-I), and eliminated the reduction of KCa and KC-I observed for phosphorylated-cTnI(WT). In isolated myofibrils, cTnI(P83S) maintained maximal tension (TMAX) and Ca(2+) sensitivity of tension (pCa50). For cTnI(WT) myofibrils, PKA-mediated phosphorylation decreased pCa50 and sped up the slow-phase relaxation (especially for those Ca(2+) conditions that heart performs in vivo). Those effects were blunted for cTnI(P83S) myofibrils. Molecular-dynamics simulations suggested cTnI(P83S) moderately inhibited an intrasubunit interaction formation between inhibitory-peptide and N-terminus, but this "blunting" effect was weaker than that with cTnI(R146G) or cTnI(R21C). In summary, cTnI(P83S) has similar effects as other HCM-associated cTnI mutations on troponin and myofibril function even though it is in the I-T arm of cTnI. PMID:27150586

  20. microRNA-208a in an early stage myocardial infarction rat model and the effect on cAMP-PKA signaling pathway

    PubMed Cen