A genetic variation in the adenosine A2A receptor gene (ADORA2A) contributes to individual sensitivity to caffeine effects on sleep.

Caffeine is the most widely used stimulant in Western countries. Some people voluntarily reduce caffeine consumption because it impairs the quality of their sleep. Studies in mice revealed that the disruption of sleep after caffeine is mediated by blockade of adenosine A2A receptors. Here we show in humans that (1) habitual caffeine consumption is associated with reduced sleep quality in self-rated caffeine-sensitive individuals, but not in caffeine-insensitive individuals; (2) the distribution of distinct c.1083T>C genotypes of the adenosine A2A receptor gene (ADORA2A) differs between caffeine-sensitive and -insensitive adults; and (3) the ADORA2A c.1083T>C genotype determines how closely the caffeine-induced changes in brain electrical activity during sleep resemble the alterations observed in patients with insomnia. These data demonstrate a role of adenosine A2A receptors for sleep in humans, and suggest that a common variation in ADORA2A contributes to subjective and objective responses to caffeine on sleep.

A structural comparison of molybdenum cofactor-containing enzymes.

This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes. Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed. This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus. On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.

Serine-202 is the putative precursor of the active site dehydroalanine of phenylalanine ammonia lyase. Site-directed mutagenesis studies on the enzyme from parsley (Petroselinum crispum L.).

To investigate the possible role of serine as a precursor of dehydroalanine at the active site of phenylalanine ammonia lyase, two serines, conserved in all known PAL and histidase sequences, were changed to alanine by site-directed mutagenesis. The resulting mutant genes were subcloned into the expression vector pT7.7 and the gene products were assayed for PAL activity. Mutant PALMutS209A showed the same catalytic property as wild-type PAL, whereas mutant PALMutS202A was devoid of catalytic activity, indicating that serine-202 is the most likely precursor of the active site dehydroalanine.

Isolation, sequencing and expression in E. coli of the urocanase gene from white clover (Trifolium repens).

The urocanase gene was detected in a clone obtained from a genomic library of white clover. The entire gene has been sequenced and expressed in the pT7-7/E. coli BL 21 (DE 3) system. The deduced sequence of the plant urocanase is 72% homologous with that of the well-characterized urocanase from Pseudomonas putida. The purification procedure, as well as kinetic and electrophoretic behaviour, of the new enzyme are described.

The role and source of 5'-deoxyadenosyl radical in a carbon skeleton rearrangement catalyzed by a plant enzyme.

The last step in the biosynthesis of tropane alkaloids is the carbon skeleton rearrangement of littorine to hyoscyamine. The reaction is catalyzed by a cell-free extract prepared from cultured hairy roots of Datura stramonium. Adenosylmethionine stimulated the rearrangement 10-20-fold and showed saturation kinetics with an apparent Km of 25 microM. It is proposed that S-adenosylmethionine is the source of a 5'-deoxyadenosyl radical which initiates the rearrangement in a similar manner as it does in analogous rearrangements catalyzed by coenzyme B12-dependent enzymes. Possible roles of S-adenosylmethionine as a radical source in higher plants are discussed.

Cloning and sequencing the urocanase gene (hutU) from Pseudomonas putida.

A clone harbouring the entire urocanase gene (hutU) was obtained from a genomic library of Pseudomonas putida using oligonucleotide probes synthesised on the basis of known flanking sequences. One subunit of urocanase consists of 556 amino acids and has a molecular mass of 60,771 Da.

Sleep and rest facilitate auditory learning.

Sleep is superior to waking for promoting performance improvements between sessions of visual perceptual and motor learning tasks. Few studies have investigated possible effects of sleep on auditory learning. A key issue is whether sleep specifically promotes learning, or whether restful waking yields similar benefits. According to the "interference hypothesis," sleep facilitates learning because it prevents interference from ongoing sensory input, learning and other cognitive activities that normally occur during waking. We tested this hypothesis by comparing effects of sleep, busy waking (watching a film) and restful waking (lying in the dark) on auditory tone sequence learning. Consistent with recent findings for human language learning, we found that compared with busy waking, sleep between sessions of auditory tone sequence learning enhanced performance improvements. Restful waking provided similar benefits, as predicted based on the interference hypothesis. These findings indicate that physiological, behavioral and environmental conditions that accompany restful waking are sufficient to facilitate learning and may contribute to the facilitation of learning that occurs during sleep.

Evidence for a 1,2 Shift of a Hydrogen Atom in a Radical Intermediate of the Methylmalonyl-CoA Mutase Reaction.

An excellent substrate of methylmalonyl-CoA mutase, methylmalonyl-carba-(dethia) coenzyme A (methylmalonyl-CH(2)-CoA), was synthesized by a chemoenzymatic method and its alpha-proton was exchanged with deuterium by long-term incubation in deuterium oxide at pH 6.9. After addition of highly purified epimerase-free methylmalonyl-CoA mutase the enzymatic rearrangement was monitored by 1H NMR spectroscopy. Already in the initial phases of the reaction only 72% of the produced succinyl-CH(2)-CoA was monodeuterated, while unlabeled and geminally dideuterated species, 14% of each, were also formed. After the addition of more enzyme the equilibrium (methylmalonyl-CoA:succinyl-CoA = 1:20) was quickly established, while the proportion of unlabeled succinyl-CH(2)-CoA rose to 30% and the geminally dideuterated species were slowly transformed to vicinally dideuterated ones. After 19 h of incubation the ratio of the unlabeled, monodeuterated, and dideuterated species was roughly 1:1:1 while no appreciable deuterium incorporation from the solvent occurred. The unexpected disproportionation of deuterium can be best explained by a 1,2 shift of a hydrogen atom in the succinyl-CH(2)-CoA radical intermediate competing with the hydrogen transfer from 5'-deoxyadenosine. A precedence for such a hydrogen shift in a radical was previously observed only in the mass spectrometer and was supported by ab initio calculations. Copyright 2000 Academic Press.

The use of carba(dethia)-coenzyme A (CH2CoA) derivatives for mechanistic investigations.

A novel class of acyl-coenzyme A analogues has been synthesized in which the sulphur atom is replaced by methylene. In contrast to their natural thiolester counterparts these acyl-CH2CoA analogues are stable to hydrolysis. They are good substrates for several enzymes that do not attack the thiolester group (carboxylases, mutases, dehydrogenases, epimerases, etc.) and potent inhibitors for most enzymes that do so. Some of the new insights gained by the use of acyl-CH2CoA are discussed in terms of enzymatic mechanisms.

Proposal for the mechanism of action of urocanase. Inference from the inhibition by 2-methylurocanate.

Incubation of urocanase with 2-methylurocanate leads, after an initial normal reaction, to a time dependent inactivation of the enzyme. It is suggested that a tautomeric form of the product, 2-methyl-imidazolone propionate, is the actual inhibitor. On the basis of these and of published experimental data a novel mechanism is proposed for the urocanase reaction. The crucial and initial step is the electrophilic addition of enzyme-bound NAD to the 2-position of the imidazole nucleus of urocanate.

Polymorphisms of ADORA2A modulate psychomotor vigilance and the effects of caffeine on neurobehavioural performance and sleep EEG after sleep deprivation.

BACKGROUND AND PURPOSE: Prolonged wakefulness impairs sustained vigilant attention, measured with the psychomotor vigilance task (PVT), and induces a compensatory increase in sleep intensity in recovery sleep, quantified by slow-wave activity (SWA) in the sleep electroencephalogram (EEG). These effects of sleep deprivation are counteracted by the adenosine receptor antagonist caffeine, implying involvement of the adenosine neuromodulator/receptor system. To examine a role for adenosine A(2A) receptors, we investigated whether variation of the A(2A) receptor gene (ADORA2A) modified effects of caffeine on PVT and SWA after sleep deprivation. EXPERIMENTAL APPROACH: A haplotype analysis of eight single-nucleotide polymorphisms of ADORA2A was performed in 82 volunteers. In 45 young men carrying five different allele combinations, we investigated the effects of prolonged waking and 2 × 200 mg caffeine or 2 × 100 mg modafinil on psychomotor vigilance, sleepiness, and the waking and sleep EEG. KEY RESULTS: Throughout extended wakefulness, the carriers of haplotype HT4 performed faster on the PVT than carriers of non-HT4 haplotype alleles. In haplotype HT4, caffeine failed to counteract the waking-induced impairment of PVT performance and the rebound of SWA in recovery sleep. However, caffeine was effective in non-HT4 allele carriers, and modafinil reduced the consequences of prolonged waking, independently of ADORA2A haplotype. CONCLUSIONS AND IMPLICATIONS: Common genetic variation of ADORA2A is an important determinant of psychomotor vigilance in rested and sleep-deprived state. It also modulates individual responses to caffeine after sleep deprivation. These findings demonstrate a role for adenosine A(2A) receptors in the effects of prolonged wakefulness on vigilant attention and the sleep EEG.