Sex differences in NMDA receptor expression in human alcoholics.

AIM: The aim of this study was to assess whether chronic alcohol misuse affects N-methyl-d-aspartate (NMDA) receptor subunit concentrations in human cases, and whether male and female subjects respond differently. METHODS: Real-time RT-PCR normalized to GAPDH was used to assay NR1, NR2A and NR2B subunit mRNA in superior frontal (SFC) and primary motor (PMC) cortex tissue obtained at autopsy from chronic alcoholics with and without comorbid cirrhosis of the liver, and from matched controls. RESULTS: The expression of all three subunits was significantly lower in both areas of cirrhotic alcoholics than in either controls or alcoholics without comorbid disease, who did not differ significantly. Values were also influenced by the subject's sex and genotype. The mu-opiate receptor C1031G polymorphism selectively modulated NMDA transcript expression in cirrhotic-alcoholic SFC, an effect that was more marked for NR1 and NR2A than for NR2B subunit transcripts. Contrasting 5HT1B genotypes affected NMDA mRNA expression differently in male and female SFC, but not PMC, in cirrhotic alcoholics. CONCLUSION: NMDA receptor subunit expression may differentially influence male and female cirrhotic alcoholics' susceptibility to brain damage.

Cortical NMDA receptor expression in human chronic alcoholism: influence of the TaqIA allele of ANKK1.

Real-time RT-PCR normalized to GAPDH was used to assay N-methyl-D-aspartate (NMDA) receptor NR1, NR2A and NR2B subunit mRNA in human autopsy cortex tissue from chronic alcoholics with and without comorbid cirrhosis of the liver and matched controls. Subunit expression was influenced by the subject's genotype. The TaqIA polymorphism selectively modulated NMDA receptor mean transcript expression in cirrhotic-alcoholic superior frontal cortex, in diametrically opposite ways in male and female subjects. Genetic make-up may differentially influence vulnerability to brain damage by altering the excitation: inhibition balance, particularly in alcoholics with comorbid cirrhosis of the liver. The TaqIA polymorphism occurs within the poorly characterised ankyrin-repeat containing kinase 1 (ANKK1) gene. Using PCR, ANKK1 mRNA transcript was detected in inferior temporal, occipital, superior frontal and primary motor cortex of control human brain. ANKK1 expression may mediate the influence of the TaqIA polymorphism on phenotype.

The expression of NMDA receptor subunit mRNA in human chronic alcoholics.

Ethanol is a modulator at the N-methyl-d-aspartate class of glutamate receptors in the brain. In animal studies the receptor adapts to sustained ethanol exposure through altered expression of the subunits that make up the receptor complex. We used real-time RT-PCR normalized to GAPDH to assay NR1, NR2A, and NR2B subunit mRNA in superior frontal and primary motor cortex tissue obtained at autopsy from chronic alcoholics with and without co-morbid cirrhosis of the liver, and from matched controls. The expression of all three subunits was significantly lower in both areas of cirrhotic alcoholics than in the corresponding areas in both controls and alcoholics without co-morbid disease, who did not differ significantly from each other. The decrease was area-dependent when cases were partitioned by the 5-HTTLPR allele. Thus, polymorphisms in one gene can have a significant effect on the expression of a second, unrelated, gene. The expression of the N-methyl-d-aspartate glutamate receptor complex is under multifactorial control.

Spectroscopic and kinetic studies of Y114F and W116F mutants of Me2SO reductase from Rhodobacter capsulatus.

Mutants of the active site residues Trp-116 and Tyr-114 of the molybdenum-containing Me(2)SO reductase from Rhodobacter capsulatus have been examined spectroscopically and kinetically. The Y114F mutant has an increased rate constant for oxygen atom transfer from Me(2)SO to reduced enzyme, the result of lower stability of the E(red).Me(2)SO complex. The absorption spectrum of this species (but not that of either oxidized or reduced enzyme) is significantly perturbed in the mutant relative to wild-type enzyme, consistent with Tyr-114 interacting with bound Me(2)SO. The as-isolated W116F mutant is only five-coordinate, with one of the two equivalents of the pyranopterin cofactor found in the enzyme dissociated from the molybdenum and replaced by a second Mo=O group. Reduction of the mutant with sodium dithionite and reoxidation with Me(2)SO, however, regenerates the long-wavelength absorbance of functional enzyme, although the wavelength maximum is shifted to 670 nm from the 720 nm of wild-type enzyme. This "redox-cycled" mutant exhibits a Me(2)SO reducing activity and overall reaction mechanism similar to that of wild-type enzyme but rapidly reverts to the inactive five-coordinate form in the course of turnover.

A multicopper oxidase is essential for manganese oxidation and laccase-like activity in Pedomicrobium sp. ACM 3067.

Pedomicrobium sp. ACM 3067 is a budding-hyphal bacterium belonging to the alpha-Proteobacteria which is able to oxidize soluble Mn2+ to insoluble manganese oxide. A cosmid, from a whole-genome library, containing the putative genes responsible for manganese oxidation was identified and a primer-walking approach yielded 4350 bp of novel sequence. Analysis of this sequence showed the presence of a predicted three-gene operon, moxCBA. The moxA gene product showed homology to multicopper oxidases (MCOs) and contained the characteristic four copper-binding motifs (A, B, C and D) common to MCOs. An insertion mutation of moxA showed that this gene was essential for both manganese oxidation and laccase-like activity. The moxB gene product showed homology to a family of outer membrane proteins which are essential for Type I secretion in Gram-negative bacteria. moxBA has not been observed in other manganese-oxidizing bacteria but homologues were identified in the genomes of several bacteria including Sinorhizobium meliloti 1021 and Agrobacterium tumefaciens C58. These results suggest that moxBA and its homologues constitute a family of genes encoding an MCO and a predicted component of the Type I secretion system.

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus.

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo(VI) form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo(VI) form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo(VI) species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion.

Site-directed mutagenesis of dimethyl sulfoxide reductase from Rhodobacter capsulatus: characterization of a Y114 --> F mutant.

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.

The first non-turnover voltammetric response from a molybdenum enzyme: direct electrochemistry of dimethylsulfoxide reductase from Rhodobacter capsulatus.

The first direct voltammetric response from a molybdenum enzyme under non-turnover conditions is reported. Cyclic voltammetry of dimethylsulfoxide reductase from Rhodobacter capsulatus reveals a reversible Mo(VI/V) response at +161 mV followed by a reversible Mo(V/IV) response at -102 mV versus NHE at pH 8. The higher potential couple exhibits a pH dependence consistent with protonation upon reduction to the Mo(V) state and we have determined the p K(a) for this semi-reduced species to be 9.0. The lower potential couple is pH independent within the range 5