Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer's disease.

Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na(+)-dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V(max), without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.

Structure of the adenylation domain of an NAD+-dependent DNA ligase

Background: DNA ligases catalyse phosphodiester bond formation between adjacent bases in nicked DNA, thereby sealing the nick. A key step in the catalytic mechanism is the formation of an adenylated DNA intermediate. The adenyl group is derived from either ATP (in eucaryotes and archaea) or NAD+4 (in bacteria). This difference in cofactor specificity suggests that DNA ligase may be a useful antibiotic target.

Results: The crystal structure of the adenylation domain of the NAD+-dependent DNA ligase from Bacillus stearothermophilus has been determined at 2.8 Å resolution. Despite a complete lack of detectable sequence similarity, the fold of the central core of this domain shares homology with the equivalent region of ATP-dependent DNA ligases, providing strong evidence for the location of the NAD+-binding site.

Conclusions: Comparison of the structure of the NAD+4-dependent DNA ligase with that of ATP-dependent ligases and mRNA-capping enzymes demonstrates the manifold utilisation of a conserved nucleotidyltransferase domain within this family of enzymes. Whilst this conserved core domain retains a common mode of nucleotide binding and activation, it is the additional domains at the N terminus and/or the C terminus that provide the alternative specificities and functionalities in the different members of this enzyme superfamily.

Electrochemical ammonia gas sensing in nonaqueous systems: A comparison of propylene carbonate with room temperature ffonc liquids

First, the direct and indirect electrochemical oxidation of ammonia has been studied by cyclic voltammetry at glassy carbon electrodes in propylene carbonate. In the case of the indirect oxidation of ammonia, its analytical utility of indirect for ammonia sensing was examined in the range from 10 and 100 ppm by measuring the peak current of new wave resulting from reaction between ammonia and hydroquinone, as function of ammonia concentration, giving a sensitivity 1.29 x 10(-7) A ppm(-1) (r(2)=0.999) and limit-of-detection 5 ppm ammonia. Further, the direct oxidation of ammonia has been investigated in several room temperature ionic liquids (RTILs), namely 1-butyl-3-methylimidazolium tetrafluoroborate ([C(4)mim] [BF4]), 1-butyl-3-methylimiclazolium trifluoromethylsulfonate ([C4mim] [OTf]), 1-Ethyl -3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C(2)mim] [NTf2]), 1-butyl-3-methylimidazolium bis(tritluoromethylsulfonyl)imide ([C4mim] [NTf2]) and 1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim] [PF6]) on a 10 put diameter Pt microdisk electrode. In four of the RTILs studied, the cyclic voltammetric analysis suggests that ammonia is initially oxidized to nitrogen, N-2, and protons, which are transferred to an ammonia molecule, forming NH4+ via the protonation of the anion(s) (A(-)). However, in [C4mim] [PF6], the protonated anion was formed first, followed by NH4+. In all five RTILs, both HA and NH4+ are reduced at the electrode surface, forming hydrogen gas, which is then oxidized. The analytical ability of this work has also been explored further, giving a limit-of-detection close to 50 ppm in [C(2)mim] [NTf2], [C(4)mim] [OTf], [C(4)mim] [BF4], with a sensitivity of ca. 6 x 10(-7) A ppm(-1) (r(2) = 0.999) for all three ionic liquids, showing that the limit of detection was ca. ten times larger than that in propylene carbonate since ammonia in propylene carbonate might be more soluble in comparison with RTILs when considering the higher viscosity of RTILs.

Mechanistic studies of the electro-oxidation pathway of ammonia in several room-temperature ionic liquids

A mechanistic study of the direct oxidation of ammonia has been reported in several room-temperature ionic liquids (RTILs), namely, [C(4)mim][BF4], [C(4)mim][OTf], [C(2)mim][NTf2], [C(4)mim][NTf2], and [C(4)mim][PF6], on a 10 mu m diameter Pt microdisk electrode. In four of the RTILs studied, the cyclic voltammetric analysis suggests that ammonia is initially oxidized to nitrogen, N-2, and protons, which are transferred to an ammonia molecule, forming NH4+ via the protonation of the anion(s) (A(-)). In contrast, NH4+ is formed first in [C(4)mim][PF6], followed by the protonated anion(s), HA. In all five RTILs, both HA and NH4+ are reduced at the electrode surface, forming hydrogen gas, which is then oxidized. The effect of changing the RTIL anion is discussed, and this may have implications in the defining of pK(a) in RTIL media. This work also has implications in the possible amperometric sensing of ammonia gas.

Elucidation of the electrochemical oxidation pathway of ammonia in dimethylformamide and the room temperature ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide

The direct electrochemical oxidation of ammonia has been examined in both the organic solvent dimethylformamide (DMF) and the room temperature ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide [EMIM][N(Tf)(2)]. The corresponding voltammetric responses have been shown to be similar in each solvent with a broad oxidative wave occurring upon the introduction of ammonia to the solution and the appearance of a new reductive wave following the oxidation. The oxidative reaction process has been examined and a suitable reaction pathway has been deduced, corresponding to the formation of ammonium cations after oxidation of the ammonia. A linear response of limiting current against vol% ammonia was observed in both DMF and [EMIM][N(Tf)(2)], suggesting potential application for analytical methods.

Determination of ammonia based on the electro-oxidation of hydroquinone in dimethylformamide or in the room temperature ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide

The results detail a novel methodology for the electrochemical determination of ammonia based on its interaction with hydroquinone in DMF. It has been shown that ammonia reversibly removes protons from the hydroquinone molecules, thus facilitating the oxidative process with the emergence of a new wave at less positive potentials. The analytical utility of the proposed methodology has been examined with a linear range from 10 to 95 ppm and corresponding limit-of-detection of 4.2 ppm achievable. Finally, the response of hydroquinone in the presence of ammonia has been examined in the room temperature ionic liquid 1-ethyl-3-methylimidazolium bis(trifluormethylsulfonyl)imide, [EMIM][N(Tf)(2)]. Analogous voltammetric waveshapes to that observed in DMF were obtained, thereby confirming the viability of the method in either DMF or [EMIM][N(Tf)(2)] as solvent. (C) 2003 Elsevier B.V. All rights reserved.

Insight into the adsorption competition and the relationship between dissociation and association reactions in ammonia synthesis

Ammonia synthesis on three metal surfaces (Zr, Ru, and Pd) is investigated using density functional theory calculations. In addition to N-2 dissociation, all the transition states of the hydrogenation reactions from N to NH3 are located and the reaction energy profiles at both low and high surface coverages are compared and analyzed. The following are found: (i) Surface coverage effect on dissociation reactions is more significant than that on association reactions. (ii) The difference between N and H chemisorption energies, the so-called chemisorption energy gap which is a measure of adsorption competition, is vital to the reactivity of the catalysts. (iii) The hydrogenation barriers can considerably affect the overall rate of ammonia synthesis. A simple model to describe the relationship between dissociation and association reactions is proposed. (c) 2007 American Institute of Physics.

A density functional theory study of stepwise addition reactions in ammonia synthesis on Ru(0001)

To shed light on stepwise addition reactions in ammonia synthesis, density functional theory calculations are carried out to investigate NHx (x = 1-3) formation on Ru(0001). The reactions on a flat surface are first examined. Transition states and reaction barriers are determined. It is found that the reaction barriers for these stepwise addition reactions are rather high. For example, the barrier for NH hydrogenation is calculated to be 1.28 eV, which is comparable with that of N-2 dissociation. One of the stepwise addition reactions, NH + H --> NH2, on a stepped surface is also considered. Interestingly, the reaction barrier is found to be significantly lower than that on the flat surface, but is considerably higher than that of N-2 dissociation on the same stepped surface. In addition, the coverage effect on the reaction energetics is also addressed. (C) 2001 Published by Elsevier Science B.V.

Stepwise addition reactions in ammonia synthesis: A first principles study

Catalytic ammonia synthesis is believed to proceed via dissociation of N-2 and H-2 with subsequent stepwise addition reactions from an adsorbed nitrogen atom to NH3. The first step, N-2 dissociation, has been thoroughly studied. However, little is known about the microscopic details of the stepwise addition reactions. To shed light on these stepwise addition reactions, density functional theory calculations with the generalized gradient approximation are employed to investigate NHx (x=1,3) formation on Ru(0001). Transition states and reaction barriers are determined in each elementary step. It is found that the reaction barriers for stepwise addition reactions are rather high, for example, the barrier for NH hydrogenation is calculated to be 1.28 eV, which is comparable with that of N-2 dissociation. In addition, one of the stepwise addition reactions on a stepped surface is also considered. The reaction barrier is found to be much higher than that of N-2 dissociation on the same stepped surface, which indicates the importance of stepwise addition reactions in ammonia synthesis. (C) 2001 American Institute of Physics.