Turnover rate of coenzyme A in mouse brain and liver.

Coenzyme A (CoA) is a fundamental cofactor involved in a number of important biochemical reactions in the cell. Altered CoA metabolism results in severe conditions such as pantothenate kinase-associated neurodegeneration (PKAN) in which a reduction of the activity of pantothenate kinase isoform 2 (PANK2) present in CoA biosynthesis in the brain consequently lowers the level of CoA in this organ. In order to develop a new drug aimed at restoring the sufficient amount of CoA in the brain of PKAN patients, we looked at its turnover. We report here the results of two experiments that enabled us to measure the half-life of pantothenic acid, free CoA (CoASH) and acetylCoA in the brains and livers of male and female C57BL/6N mice, and total CoA in the brains of male mice. We administered (intrastriatally or orally) a single dose of a [13C3-15N-18O]-labelled coenzyme A precursor (fosmetpantotenate or [13C3-15N]-pantothenic acid) to the mice and measured, by liquid chromatography-mass spectrometry, unlabelled- and labelled-coenzyme A species appearance and disappearance over time. We found that the turnover of all metabolites was faster in the liver than in the brain in both genders with no evident gender difference observed. In the oral study, the CoASH half-life was: 69 ± 5 h (male) and 82 ± 6 h (female) in the liver; 136 ± 14 h (male) and 144 ± 12 h (female) in the brain. AcetylCoA half-life was 74 ± 9 h (male) and 71 ± 7 h (female) in the liver; 117 ± 13 h (male) and 158 ± 23 (female) in the brain. These results were in accordance with the corresponding values obtained after intrastriatal infusion of labelled-fosmetpantotenate (CoASH 124 ± 13 h, acetylCoA 117 ± 11 and total CoA 144 ± 17 in male brain).

A ten fold reduction of nicotine yield in tobacco smoke does not spare the central cholinergic system in adolescent mice.

The tobacco industry has gradually decreased nicotine content in cigarette smoke but the impact of this reduction on health is still controversial. Since the central cholinergic system is the primary site of action of nicotine, here, we investigated the effects of exposure of adolescent mice to tobacco smoke containing either high or low levels of nicotine on the central cholinergic system and the effects associated with cessation of exposure. From postnatal day (PN) 30 to 45, male and female Swiss mice were exposed to tobacco smoke (whole body exposure, 8h/day, 7 days/week) generated from 2R1F (HighNic group: 1.74mg nicotine/cigarette) or 4A1 (LowNic group: 0.14mg nicotine/cigarette) research cigarettes, whereas control mice were exposed to ambient air. Cholinergic biomarkers were assessed in the cerebral cortex and midbrain by the end of exposure (PN45), at short- (PN50) and long-term (PN75) deprivation. In the cortex, nicotinic cholinergic receptor upregulation was observed with either type of cigarette. In the midbrain, upregulation was detected only in HighNic mice and remained significant in females at short-term deprivation. The high-affinity choline transporter was reduced in the cortex: of HighNic mice by the end of exposure; of both HighNic and LowNic females at short-term deprivation; of LowNic mice at long-term deprivation. These decrements were separable from effects on choline acetyltransferase and acetylcholinesterase activities, suggesting cholinergic synaptic impairment. Here, we demonstrated central cholinergic alterations in an animal model of tobacco smoke exposure during adolescence. This system was sensitive even to tobacco smoke with very low nicotine content.

Occurrence and expression of tricarboxylate synthases in Ralstonia eutropha.

2-Methylcitrate synthase (2-MCS1) and citrate synthase (CS) of Ralstonia eutropha strain H16 were separated by affinity chromatography and analyzed for their substrate specificities. 2-MCS1 used not only the primary substrate propionyl-CoA but also acetyl-CoA and, at a low rate, even butyryl-CoA and valeryl-CoA for condensation with oxaloacetate. The KM values for propionyl-CoA and acetyl-CoA were 0.061 or 0.35 mM, respectively. This enzyme is therefore a competitor for acetyl-CoA during biosynthesis of poly(3-hydroxybutyrate) (PHB) and has to be taken into account if metabolic fluxes are calculated for PHB biosynthesis. In contrast, CS could not use propionyl-CoA as a substrate. The gene-encoding CS (cisY) of R. eutropha was cloned and encodes for a protein consisting of 433 amino acids with a calculated molecular weight of 48,600 Da; it is not truncated in the N-terminal region. Furthermore, a gene encoding a second functionally active 2-methylcitrate synthase (2-MCS2, prpC2) was identified in the genome of R. eutropha. The latter was localized in a gene cluster with genes for an NAD(H)-dependent malate dehydrogenase and a putative citrate lyase. RT-PCR analysis of R. eutropha growing on different carbon sources revealed the transcription of prpC2. In addition, cells of recombinant Escherichia coli strains harboring prpC2 of R. eutropha exhibited high 2-MCS activity of 0.544 U mg-1. A prpC2 knockout mutant of R. eutropha exhibited an identical phenotype as the wild type if grown on different media. 2-MCS2 seems to be dispensable, and a function could not be revealed for this enzyme.

Chlorpyrifos exposure during neurulation: cholinergic synaptic dysfunction and cellular alterations in brain regions at adolescence and adulthood.

The developmental neurotoxicity of chlorpyrifos (CPF) involves multiple mechanisms, thus rendering the immature brain susceptible to adverse effects over a wide window of vulnerability. Earlier work indicated that CPF exposure at the neural tube stage elicits apoptosis and disrupts mitotic patterns in the brain primordium but that rapid recovery ensues before birth. In the current study, we assessed whether defects in cholinergic synaptic activity emerge later in development. CPF was given to pregnant rats on gestational days 9-12, using regimens devoid of overt maternal or fetal toxicity. We then examined subsequent development of acetylcholine systems and compared the effects to those on general biomarkers of cell development. Choline acetyltransferase (ChAT), a constitutive marker for cholinergic nerve terminals, was increased in the hippocampus and striatum in adolescence and adulthood. In contrast, hemicholinium-3 (HC-3) binding to the presynaptic choline transporter, an index of nerve impulse activity, was markedly subnormal. Furthermore, m2-muscarinic cholinergic receptor binding was significantly reduced, instead of showing the expected compensatory upregulation for reduced neural input. CPF also elicited delayed-onset alterations in biomarkers of cell packing density, cell number, cell size and neuritic projections, involving brain regions both with and without reductions in indices of cholinergic activity. In combination with earlier results, the current findings indicate that the developing brain, and especially the hippocampus, is adversely affected by CPF regardless of whether exposure occurs early or late in brain development, and that defects emerge in adolescence or adulthood even in situations where normative values are initially restored in the immediate post-exposure period.

Postnatal development of choline acetyltransferase activity in the rat laterodorsal tegmental nucleus.

Cholinergic neurons in the laterodorsal tegmental nucleus (LDT) have important roles in the regulation of sleep or waking in adult animals. In neonatal animals, sleep is largely occupied by paradoxical sleep. To investigate the relation between the cholinergic neurons in the LDT and the development of neonatal sleep, we dissected the LDT of rat by micropunch method at postnatal day 1--45 and measured the activity of choline acetyltransferase (ChAT). Either specific or total activity of ChAT was weak in the first week, increased strikingly in the second week and then moderately thereafter. The time course of the increase in ChAT activity correlates well to that of the decrease in the amount of paradoxical sleep or body twitches after birth.

Synthesis of O-[11C]Acetyl CoA, O-[11C]Acetyl-L-carnitine, and L-[11C]carnitine labelled in specific positions, applied in PET studies on rhesus monkey.

The syntheses of L-carnitine, O-acetyl CoA, and O-acetyl-L-carnitine labelled with 11C at the 1- or 2-position of the acetyl group or the N-methyl position of carnitine, using the enzymes acetyl CoA synthetase and carnitine acetyltransferase, are described. With a total synthesis time of 45 min, O-[1-11C]acetyl CoA and O-[2[11C]acetyl CoA was obtained in 60-70% decay-corrected radiochemical yield, and O-[1-11C]acetyl-L-carnitine and O-[2-11C] acetyl-L-carnitine in 70-80% yield, based on [1-11C]acetate or [2-11C]acetate, respectively. By an N-methylation reaction with [11C]methyl iodide, L-[methyl-11C]carnitine was obtained within 30 min, and O-acetyl-L-[methyl-11C]carnitine within 40 min, giving a decay-corrected radiochemical yield of 60% and 40-50%, respectively, based on [11C]methyl iodide. Initial data of the kinetics of the different 11C-labelled L-carnitine and acetyl-L-carnitines in renal cortex of anaesthetized monkey (Macaca mulatta) are presented.