Showing papers on "Monoamine oxidase B published in 1998"
TL;DR: A new polymorphism upstream of the gene for monoamine oxidase A, which consists of a 30-bp repeated sequence present in 3, 3.5, 4, or 5 copies, may be useful as both a functional and an anonymous genetic marker for MAOA.
Abstract: We describe a new polymorphism upstream of the gene for monoamine oxidase A (MAOA), an important enzyme in human physiology and behavior. The polymorphism, which is located 1.2 kb upstream of the MAOA coding sequences, consists of a 30-bp repeated sequence present in 3, 3.5, 4, or 5 copies. The polymorphism is in linkage disequilibrium with other MAOA and MAOB gene markers and displays significant variations in allele frequencies across ethnic groups. The polymorphism has been shown to affect the transcriptional activity of the MAOA gene promoter by gene fusion and transfection experiments involving three different cell types. Alleles with 3.5 or 4 copies of the repeat sequence are transcribed 2–10 times more efficiently than those with 3 or 5 copies of the repeat, suggesting an optimal length for the regulatory region. This promoter region polymorphism may be useful as both a functional and an anonymous genetic marker for MAOA.
1,082 citations
TL;DR: Dietary compounds can attenuate peroxide production in glial cells by either inhibiting the deamination of monoamines or acting as a free radical scavenger.
Abstract: Glial cell monoamine oxidase (MAO) activity has been implicated as a contributor to oxidative neuronal damage associated with various neurodegenerative diseases. The attenuation of MAO activity may provide protection against oxidative neurodegeneration. In this investigation, the presence of MAO-B in rat C6 astrocyte cells was substantiated by dose-dependent inhibition of enzyme activity in the presence of chlorgyline-HCl and L-deprenyl. The present study evaluated various dietary-derived food constituents for evidence of inhibition on oxidative deamination of monoamines or peroxide radical trapping capacity. Results of this investigation indicate that compounds which inhibit C6 glial cell MAO enzyme activity or scavenge peroxide product include chlorogenic acid, (+)-catechin, taxifolin, (-)-epigallocatechin gallate (EGCG), fisetin, coenzyme Q0, curcumin, sesamol, morin, sesame oil, silymarin, green tea, ferulic acid, caffeic acid, and rutin hydrate. The results of this study indicate that dietary compounds can attenuate peroxide production in glial cells by either inhibiting the deamination of monoamines or acting as a free radical scavenger.
126 citations
TL;DR: Though nicotine is at the core of the neuropharmacological actions of tobacco smoke, MAO B inhibition may also be an important variable in understanding and treating tobacco smoke addiction.
Abstract: We measured the concentration of brain monoamine oxidase B (MAO B; EC 1.4.3.4) in 8 smokers and compared it with that in 8 non-smokers and in 4 former smokers using positron emission tomography (PET) and deuterium substituted [11C]L-deprenyl ([11C]L-deprenyl-D2) as a radiotracer for MAO B. Smokers had significantly lower brain MAO B than non-smokers as measured by the model term lambda k3 which is a function of MAO B activity. Reductions were observed in all brain regions. Low brain MAO B in the cigarette smoker appears to be a pharmacological rather than a genetic effect since former smokers did not differ from non-smokers. Brain MAO B inhibition by cigarette smoke is of relevance in light of the inverse association between smoking and Parkinson's disease and a high prevalence of smoking in psychiatric disorders and in substance abuse. Though nicotine is at the core of the neuropharmacological actions of tobacco smoke, MAO B inhibition may also be an important variable in understanding and treating tobacco smoke addiction.
120 citations
TL;DR: Striatal extracellular fluid concentrations of dopamine and metabolites in response to direct striatal administration of two L-DOPA boluses administered sequentially were determined in three rhesus monkeys during halothane anesthesia.
Abstract: Striatal extracellular fluid concentrations of dopamine and metabolites in response to direct striatal administration of two L-DOPA boluses administered sequentially were determined in three rhesus monkeys during halothane anesthesia. Whereas in an initial microdialysis run, generation of dopamine was less following the second L-DOPA bolus than the first, in a subsequent run, in which the selective MAO-B inhibitor R(+)-Npropargyl-1-aminoindan (rasagiline) was administered systemically (0.2 mg/ kg s.c.) between the two L-DOPA boluses, generation of dopamine was greater following the second bolus.
105 citations
TL;DR: The protective action of rasagiline on dopaminergic neurons, even under stringent serum-free conditions, is striking, and warrants further investigation for a role in the treatment of Parkinson's disease.
Abstract: Both deprenyl and rasagiline (R(+)-N-propargyl-1-aminoindane mesylate), at a concentration of 1-10 microM, increased survival in vitro of rat E14 mesencephalic dopaminergic neurons that had been primed with 10% serum for 12 h (p < 0.05). Rasagiline, but not deprenyl, also increased total neuronal (MAP2-positive) survival (p < 0.05) Under serum-free conditions, rasagiline, but not deprenyl, retained its neuroprotective action on dopaminergic neurones. GABAergic neurons were not affected by either deprenyl or rasagiline. Clorgyline, an MAO-A inhibitor, did not exert any of these effects. The protective action of rasagiline on dopaminergic neurons, even under stringent serum-free conditions, is striking, and warrants further investigation for a role in the treatment of Parkinson's disease.
99 citations
TL;DR: Results suggest that both (−)‐deprenyl and melatonin up‐regulate GDNF gene expression at threshold doses lower than that needed for altering MAOB activity and/or the antioxidant enzyme systems, respectively.
Abstract: Glial cell line-derived neurotrophic factor (GDNF) has been shown to be a preferentially selective neurotrophic factor for dopamine (DA) neurons. In the present study, we have examined the distribution of GDNF mRNA expression in several major DA-containing cell body and terminal areas and the regulation of GDNF mRNA expression upon various pharmacological treatments. Results indicated that there is a relatively higher GDNF mRNA level in neurons of the nigrostriatal and mesolimbic dopaminergic pathways. Upon chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment (30 mg/kg, i.p., for 7 days), DA level was decreased, whereas GDNF mRNA expression was increased in the striatum, suggesting that more GDNF is synthesized and expressed to cope with the neurotoxin insult. Furthermore, among several DA neuron protective and/or therapeutic agents examined, both intrastriatal injections of (−)-deprenyl (1.25 μg and 2.5 μg) and melatonin (30 μg, 60 μg, and 120 μg) significantly enhanced GDNF mRNA expression in the striatum, whereas the same concentrations of (−)-deprenyl did not affect monoamine oxidase B (MAOB) activity, although it increased glutathione peroxidase (GPx) and/or superoxide dismutase (SOD) activities. Similarly, the same concentrations of melatonin did not alter SOD or GPx activities, except that the highest dose of melatonin (120 μg) increased lipid peroxidation in the striatum. Conversely, GM1 ganglioside injection (45 μg) lacked of an effect on GDNF mRNA expression. Together, these results suggest that both (−)-deprenyl and melatonin up-regulate GDNF gene expression at threshold doses lower than that needed for altering MAOB activity and/or the antioxidant enzyme systems, respectively. These results provide new information on the neuroprotective and therapeutic mechanisms of (−)-deprenyl and melatonin on DA neurons. J. Neurosci. Res. 53:593–604, 1998. © 1998 Wiley-Liss, Inc.
95 citations
TL;DR: Drug tolerability data for the elderly show that moclobemide is one of the most well tolerated compounds, and published data are insufficient to be able to give a conclusive tolerability statement regarding the use of these compounds in elderly people.
Abstract: Monoamine oxidase inhibitors (MAOIs) are mainly used in psychiatry for the treatment of depressive disorders and in neurology for the treatment of Parkinson's disease. While the classical, nonselective and nonreversible MAOIs, such as phenelzine and tranylcypromine, are characterised by the risk of inducing a hypertensive crisis when dietary tyramine is ingested, the selective monoamine oxidase-B (MAO-B) inhibitor selegiline (deprenyl) and, even more so, the selective and reversible monoamine oxidase-A (MAO-A) inhibitor moclobemide, are free from this potential interaction. Drug tolerability data for the elderly show that moclobemide is one of the most well tolerated compounds. Selegiline, especially when used in combination with levodopa, can cause anorexia, dry mouth, dyskinesia and, most problematic, orthostatic hypotension. For the traditional MAOIs, phenelzine and tranylcypromine, published data are insufficient to be able to give a conclusive tolerability statement regarding the use of these compounds in elderly people. Although orthostatic hypotension occurs in most patients treated with traditional MAOIs, the incidence in elderly patients with depression does not appear to be greater than that reported with tricyclic antidepressants.
87 citations
TL;DR: The findings, although of limited statistical precision, suggest that the G allele of this MAO-B polymorphism may relate to PD risk.
Abstract: Monoamine oxidase B (MAO-B) is an enzyme that has relevance for Parkinson disease (PD) because of its roles in catabolizing dopamine and potentially activating exogenous neurotoxicants. A polymorphism of the gene encoding MAO-B has been identified as a single base change (A or G) in intron 13 of the X chromosome. The A allele was previously associated with an approximately twofold risk of PD. The present study compared A and G allele frequencies between newly diagnosed idiopathic PD cases and a control group free of neurodegenerative diseases. All study subjects were Caucasian. Cases were 37 men and 25 women, age 37-80 years; controls were 50 men and 29 women, age 45-82 years. MAO-B genotype was determined by the allele-specific polymerase chain reaction on DNA extracted from peripheral lymphocytes. In complete contrast to previous studies, elevated risks were detected with the G allele. The age-adjusted odds ratio for the G allele in males was 1.87 ((95% confidence interval) 0.78-4.47). Among females the age-adjusted odds ratios were 5.00 ((95% confidence interval) 1.13-22.1) for the GA genotype and 5.60 ((95% confidence interval) 1.01-30.9) for the GG genotype. These findings, although of limited statistical precision, suggest that the G allele of this MAO-B polymorphism may relate to PD risk.
76 citations
TL;DR: It is suggested that estrogen exerts a tissue-specific, differential regulation of MAO-A and -B activity in brain and in other tissues as well as in heart, adrenal, lung and small intestine.
62 citations
TL;DR: It is suggested that impulsiveness is a behavioural component that may be operating independent of the other components, like hyperactivity and deficient sustained attention, and that can be reduced by chronic MAO-B inhibition with L-deprenyl in this strain of rats.
61 citations
TL;DR: Results indicate that rasagiline has potential for the treatment of Parkinson's Disease and this compound is currently under development for that indication.
Abstract: (+)-N-Propargyl-1-aminoindan (rasagiline) and a series of derivatives have been synthesized and screened for monoamine oxidase inhibitory activity. Rasagiline and several analogues were found be highly selective and potent inhibitors of the B form of the enzyme in contrast to the levorotatory enantiomer which was not active. The results indicate that rasagiline has potential for the treatment of Parkinson’s Disease. This compound is currently under development for that indication.
TL;DR: MAO-N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme, and some aspects of the substrate specificity resemble those of MAO B, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots.
Abstract: An interesting flavoprotein-type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned [Schilling, B. & Lerch, K. (1995a) Biochim. Biophys. Acta 1243, 529-537; Schilling, B. & Lerch, K. (1995b) Mol. Gen. Genet. 247, 430-438]. The properties of this MAO, as well as a substantial part of its amino acid sequence, resemble those of both MAO A and B from higher animals, raising the possibility that it may be an evolutionary precursor of these mitochondrial enzymes. It differs from MAO A and B in several respects, however, including the fact that it is soluble and of peroxisomal location and that the FAD is non-covalently attached. We have overexpressed the fungal enzyme (MAO-N) in Escherichia coli and isolated it in pure form. Since several of the observations of previous workers on MAO-N could not be reproduced, we have reexamined its substrate specificity, interaction with reversible and irreversible inhibitors and other catalytic and molecular properties. MAO-N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme. Some aspects of the substrate specificity resemble those of MAO B, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots. Contrary to a previous report [Schilling, B. & Lerch, K. (1995a) Biochim. Biophys. Acta 1243, 529-537], however, the fungal enzyme does not oxidize serotonin, norepinephrine, dopamine or other biogenic amines. MAO-N is irreversibly inhibited by stoichiometric amounts of both (-)deprenyl and clorgyline in a mechanism-based reaction, forming flavocyanine adducts with N5 of the FAD, like the mammalian enzymes, but inactivation is much faster with clorgyline than deprenyl, suggesting a closer resemblance to MAO A than B. The dissociation constants for a large number of reversible competitive inhibitors have been determined for MAO-N and comparison with similar values for MAO A and B again pointed to a greater similarity to the former than the latter.
TL;DR: It seems, that platelet MAO-B activity is influenced mainly by drug therapy, while serotonin content rather reflects clinical characteristics in dementia.
Abstract: This study aimed at determining the effect of drug therapy, age and type of dementia on biological markers Both platelet monoamine oxidase type B (MAO-B) activity and serotonin content of 57 demented patients and 20 control subjects were determined Platelet MAO-B activity was measured using [14C]tyramine as substrate Serotonin content was determined by HPLC-EC method Increased platelet serotonin content and platelet count was found in patients with dementia compared to controls A positive correlation was experienced between platelet MAO-B activity, platelet serotonin content and age Platelet MAO-B activity was higher in the haloperidol treated group, compared with patients treated with anxyolitics The main original finding of the present study is that platelet serotonin content is increased in demented patients with delusions compared to dementia without complications (p = 0006) It seems, that platelet MAO-B activity is influenced mainly by drug therapy, while serotonin content rather reflects clinical characteristics in dementia
TL;DR: The results suggest that the administration of deprenyl blocked the development of mammary tumors in part by inhibiting the metabolism of catecholamines and indoleamine and possibly by conferring a neuroprotective effect on the TIDA neurons in the MBH.
TL;DR: Novel actions of selegiline may protect neurons from ischemic or oxidative damage and suggest new therapeutic applications for L-deprenyl in vascular and neurodegenerative diseases.
Abstract: The monoamine oxidase-B inhibitor L-deprenyl (Selegiline) is effective in treating Parkinson's disease and possibly Alzheimer's disease. The neuroprotective property of L-deprenyl may be unrelated to the inhibition of monoamine oxidase-B. Since nitric oxide (NO) modulates activities including cerebral blood flow and memory, we examined the effect of L-deprenyl on NO. L-Deprenyl induced rapid increases in NO production in brain tissue and cerebral vessels. Vasodilation was produced by endothelial NO-dependent as well as NO-independent mechanisms in cerebral vessels. The drug also protected the vascular endothelium from the toxic effects of amyloid-beta peptide. These novel actions of selegiline may protect neurons from ischemic or oxidative damage and suggest new therapeutic applications for L-deprenyl in vascular and neurodegenerative diseases.
TL;DR: The first demonstration of the cellular distribution of MAO mRNA in human placenta via in situ hybridization is demonstrated and these highly specific cRNA probes can now be used to study the distribution ofMAO-A and MAO-B expression in other tissues.
Abstract: Monoamine oxidase (MAO) oxidatively deaminates vasoactive and biogenic amines and exists in two distinct forms (A and B), coded for by separate genes, which exhibit distinct substrate specificities and inhibitor sensitivities. Using specific primers for MAO-A and MAO-B mRNA in a reverse transcription-polymerase chain reaction (RT-PCR) on RNA from human liver, the predicted products for both enzymes were detected. Furthermore, RT-PCR on RNA from human placenta, believed to contain predominantly (or only) MAO-A protein, also indicated the presence of both A and B gene transcripts. The cellular distribution of MAO mRNA in placental tissue was analyzed by in situ hybridization of MAO-A and MAO-B mRNA-specific cRNA probes on paraffin sections. MAO-A mRNA was mainly evident in the syncytiotrophoblastic layer. None was detected in the vascular endothelium/smooth muscles. Significantly, MAO-B mRNA signal was also evident in the placental villi, notably in the syncytiotrophoblasts, intermediate trophoblasts, cytotrophoblasts, and the vascular endothelium. To our knowledge, this is the first demonstration of the cellular distribution of MAO mRNA in human placenta via in situ hybridization. The expression of MAO-B in placental tissue rather than in blood elements within placenta is also unequivocally demonstrated. These highly specific cRNA probes can now be used to study the distribution of MAO-A and MAO-B expression in other tissues.
TL;DR: The results indicate that Gly226 and the β-carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD.
TL;DR: In this paper, the authors performed electrophoretic mobility shift assays to investigate the pattern of protein binding to a 150 bp fragment of the proximal 5'-flanking region of the human monoamine oxidase B gene.
TL;DR: The results suggest that DA is metabolized preferentially, if not exclusively by MAO-B in some regions of the monkey brain.
Abstract: The role of monoamine oxidase (MAO) type A and B on the metabolism of dopamine (DA) in discrete regions of the monkey brain was studied. Monkeys were administered (–)-deprenyl (0.25 mg/kg) or clorgyline (1.0 mg/kg) or deprenyl and clorgyline together by intramuscular injections for 8 days. Levels of DA and its metabolites, dihydroxy phenylacetic acid (DOPAC) and homovanillic acid (HVA) were estimated in frontal cortex (FC), motor cortex (MC), occipital cortex (OC), entorhinal cortex (EC), hippocampus (HI), hypothalamus (HY), caudate nucleus (CN), globus pallidus (GP) and substantia nigra (SN). (–)-Deprenyl administration significantly increased DA levels in FC, HY, CN, GP and SN (39–87%). This was accompanied by a reduction in the levels of DOPAC (37–66%) and HVA (27–79%). Clorgyline administration resulted in MAO-A inhibition by more than 87% but failed to increase DA levels in any of the brain regions studied. Combined treatment of (–)-deprenyl and clorgyline inhibited both types of MAO by more than 90% and DA levels were increased (57–245%) in all brain regions studied with a corresponding decrease in the DOPAC (49–83%) and HVA (54–88%) levels. Our results suggest that DA is metabolized preferentially, if not exclusively by MAO-B in some regions of the monkey brain.
Journal Article•
TL;DR: The results of this investigation are interpreted to suggest that selegiline by preventing the generation of free radicals, MT isoforms by scavengingFree radicals, and neurotrophins by rescuing dopaminergic neurons are capable of attenuating oxidative stress and of providing neuro-protection in PD.
Abstract: The finding that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) elicits parkinsonism in human beings suggests that endogenous or xenobiotic neurotoxic compounds may be involved in the etiology of Parkinson's disease (PD). We have shown that cerebrospinal fluid (CSF) of newly diagnosed and drug untreated patients with PD contains a low molecular weight substance(s) which inhibits the growth and function of dopaminergic neurons in culture. In addition, selegiline in a dosage below the level that inhibits monoamine oxidase B (MAO-B), protects dopaminergic neurons in culture against toxic factor(s) present in the CSF of patients with PD, and the said effect is mediated via elaboration of brain-derived neurotrophic factor (BDNF). In view of the fact that 6-hydroxydopamine (6-OHDA) or MPTP causes parkinsonism by generating free radicals, and inducers of metallothionein (MT) isoforms avert the said neurotoxicity, we intended to learn whether MT isoforms were capable of scavenging free radicals. By employing electron spin resonance spectroscopy (ESR), we examined for the first time the free radical scavenging effects of MT-I and MT-II isoforms on four types of free radicals. Solutions of 0.15 mM of MT-I and 0.3 mM of MT-II scavenged the 1,1-diphenyl-2-picrylhydrazyl radicals completely. Furthermore, they were able to scavenge hydroxyl radicals generated in a Fenton reaction. Moreover, MT-I scavenged almost 90% of the superoxide generated by the hypoxanthine and xanthine oxidase system, while MT-II could only scavenge 40 %. By using 2,2,6,6-tetramethyl-4-piperidone as a spin-trap for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin, and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidone-1--oxyl, we observed that MT-II could scavenge 92%, while MT-I could completely scavenge all the reactive species generated. The results of this investigation are interpreted to suggest that selegiline by preventing the generation of free radicals, MT isoforms by scavenging free radicals, and neurotrophins by rescuing dopaminergic neurons are capable of attenuating oxidative stress and of providing neuroprotection in PD.
TL;DR: The experiments reported here show that both MAO A and MAO B contain a redox-active disulfide at the catalytic center, implying that MAO may be a novel type of disulfides oxidoreductase and open the way to characterizing the catalytics and chemical mechanism of the enzyme.
TL;DR: This patent update relates to monoamine oxidase inhibitors, covers the patent activity in this field for the last 3 - 5 years and focuses on the most relevant filings.
Abstract: This patent update relates to monoamine oxidase inhibitors, covers the patent activity in this field for the last 3 - 5 years and focuses on the most relevant filings. Patents are classified according to the type of MAO inhibition: selective MAO-A and MAO-B inhibitors, and mixed and reversible MAO-A and MAO-B inhibitors.
TL;DR: The main reason for the poor uptake of the fluoro compound is its lower lipophilicity as compared to the iodo compound and, to a lesser degree, its metabolism, which is similar for both compounds.
TL;DR: Results indicate that Arg-42 and Thr-45 form critical noncovalent interactions with FAD that are required for the subsequent activation of MAO B by covalent coupling of FAD.
Abstract: Monoamine oxidase B (MAO B) is an integral protein of the outer mitochondrial membrane that is involved in the deamination of vasoactive and neuroactive amines. The oxidation of these amine substrates requires the cofactor FAD, which is covalently bound to Cys-397 of human MAO B. Previously, Glu-34 and Tyr-44 of MAO B have been identified as residues which engage in noncovalent interactions with FAD that are required for subsequent covalent FAD binding and generation of catalytic activity. In this study, we have identified two additional residues, Arg-42 and Thr-45, which form noncovalent contacts with FAD that are prerequisite steps to the covalent attachment of FAD. Arg-42 and Thr-45, along with Tyr-44, comprise part of a highly conserved flavin binding sequence, RXY(T,S), that is found in other flavoproteins, several of which have well-defined X-ray crystal structures. We tested the roles of Arg-42 and Thr-45 in MAO B by constructing mutant MAO B cDNAs which encode amino acid substitutions at these res...
TL;DR: The platelet contents of monoamine oxidase (MAO-B) were analyzed in children and young adults with Prader-Willi syndrome and 18 non-PWS comparison cases, suggesting monoamine dysfunction in Prader, Willi syndrome.
Abstract: The platelet contents of monoamine oxidase (MAO-B) were analyzed in 17 children and young adults with Prader-Willi syndrome and 18 non-PWS comparison cases. MAO-B activity was significantly higher in the former group, suggesting monoamine dysfunction in Prader-Willi syndrome.
TL;DR: The estrogen receptor-mediated activation of MAO in conjunction with high catecholamine concentrations in the hamster kidney as previously reported may significantly increase the production of hydrogen peroxide and hydroxyl radicals which are postulated to contribute to tumor initiation.
TL;DR: Increase in endogenous MAO I may competitively inhibit the binding of irreversibleMAO I drugs, and may also help to mediate some mood altering effects of other drugs, or procedures such as ECT.
Abstract: Recent research on tribulin [low molecular weight endogenous inhibitory activity of monoamine oxidase (MAO)] has confirmed that its level is increased in both human urine and rat tissues by stress or anxiety, and by anxiogenic drugs. However tribulin is now known to contain several different molecules. The raised inhibitory activity in rat tissues is selective for MAO-A. There is a parallel decrease in MAO-A activity ex vivo, suggesting a possible functional effect. Increase in endogenous MAO I may competitively inhibit the binding of irreversible MAO I drugs, and may also help to mediate some mood altering effects of other drugs, or procedures such as ECT. In human urine both MAO-A I and MAO-B I have been found to be increased in mild stress. Similar findings have been made with human saliva. Selective inhibitors of MAO-A have been identified from human urine, and pig brain, but it is not yet clear to what extent they account for the MAO-A I activity increased in stress.
TL;DR: If brain tissue from MAOB knockout mice contain such "non-MAO" binding sites for radiolabelled deprenyl is investigated, no binding of L-deprenyl was observed indicating that the mother compound does not act directly via a macromolecular target.
TL;DR: The hypothesis that the MAOA gene may affect the susceptibility of individuals to PD among MAOA polymorphic loci is supported.