A putative FAD-binding domain in a distinct group of oxidases including a protein involved in plant development.

Using methods for database screening with individual protein sequences and alignment blocks, a conserved domain is delineated in a group of proteins including several FAD-dependent oxidases. Two motifs within this domain resemble phosphate-binding loops and may be directly involved in FAD binding. These motifs can be readily distinguished from previously described nucleotide-binding sites using a method for database screening with position-dependent weight matrices derived from alignment blocks. Unexpectedly, this group of known and predicted FAD-dependent oxidases includes the product of the DIMINUTO gene, which is involved in Arabidopsis development, and its homologues from man and Mycobacterium leprae.     

Inhibition of MAO-B by (−)-deprenyl alters dopamine metabolism in the macaque (Macaca facicularis) brain

The present study has examined whether MAO-B has a role in DA metabolism in the primate CNS in situ. Eleven macaques (macaca facicularis) were used in this study to examine the effects of (-)-deprenyl (1 mg/kg, i.v., 2 and 24 hours). (-)-Deprenyl administration completely and selectively blocked MAO-B activity and blocked DA metabolism in the caudate nucleus and frontal cortex. DA metabolism in the substantia nigra was not affected by MAO-B inhibition. Changes in DA metabolism were accompanied by changes in 5-hydroxytryptamine (5HT) turnover: 5-hydroxyindole acetic acid (5HIAA) levels increased in the caudate and decreased in the frontal cortex. Levels of 2-phenylethylamine (PE), a putative modulator of dopaminergic transmission, were increased by MAO-B inhibition in all three brain regions examined. It is concluded that in some regions of the primate brain, in contrast to the rat, MAO-B has an important role in DA metabolism.     

Evaluation of the Biological Activity of Several Analogs of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Several analogs of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were synthesized and screened for their capacity to be oxidized by monoamine oxidase (MAO-A or MAO-B) and their capacity to produce nigrostriatal dopaminergic neurotoxicity in mice. All of the compounds were relatively weak substrates for MAO-A but many of the compounds were found to be good substrates for MAO-B. Only three of the compounds, in addition to MPTP itself, were found to be neurotoxic. These were 1-methyl-4-cyclohexyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine and 1-methyl-4-(3'-methoxyphenyl)-1,2,3,6-tetrahydropyridine. All three of these neurotoxic compounds were found to be substrates for MAO-B; in contrast no compound was found to be neurotoxic that was not oxidized by MAO-B. The capacity of the compounds studied to be oxidized by MAO-B appears to be an important aspect of the neurotoxic process.     

Failure of Glucose and Branched-Chain Amino Acids to Normalize Brain Glucose Use in Portacaval Shunted Rats

Several abnormalities in brain and plasma amino acid concentrations caused by portacaval shunting in rats return toward normal after 4 days of intravenous infusion with either glucose or glucose with branched-chain amino acids. To assess the effect of such treatment on brain energy metabolism, regional brain glucose use was measured using [14C]glucose and autoradiography, 5 weeks after portacaval shunting. In one experiment intravenous glucose or glucose with branched-chain amino acids was given for 4 days. In a separate experiment the treatment was given orally for 2 weeks, and in addition to glucose use, brain monoamines and amino acids were measured. No other food was provided; the rats had free access to water. Normally fed shunted rats and sham-operated rats served as controls. Both types of oral treatment lowered the high concentrations of tyrosine, phenylalanine, and glutamine in plasma and brain. Glucose without amino acids normalized brain tryptophan. Levels of brain norepinephrine, 5-hydroxytryptamine (serotonin), and 5-hydroxyindoleacetic acid were significantly raised after shunting. Treatment had no effect on norepinephrine but the glucose diet brought the indoles into the normal range. In contrast, neither intravenous nor oral treatment affected brain glucose use, which remained depressed by 25-30% in all brain areas examined.     

Monoamine oxidase in the hypothalamo-hypophysial region of the teleosts,Anguilla japonica and Oryzias latipes

Summary Distribution of monoamine oxidase (MAO) was histochemically examined in the hypothalamo-hypophysial region of the eel (Anguilla japonica) and the medaka (Oryzias latipes) with a modified Glenner's tryptamine-tetrazolium method. The hypothalamic neurosecretory cells showed very weak MAO activity in their perikarya. MAO-positive fibers were present in close contact with the neurosecretory cells, suggesting that monoaminergic fibers participate in the control of neurosecretory cell activity. The nucleus lateralis tuberis (NLT) contained cells exhibiting strong MAO activity. These cells must be monoaminergic neurons.In the anterior region of the neurohypophysis of both eel and medaka, two bundles of MAO-positive fibers originating from the NLT proceed down along each side of the third ventricle into the pars distalis. This suggests that monoaminergic neurons of the NLT are involved in the release of hormones from the pars distalis. In addition to these tracts, numerous MAO-positive fibers proceed backward from the post-optic area and end around the blood capillaries located between the neurohypophysis and the pars intermedia in both species.I wish to express my gratitude to Prof. H. Kobayashi for his valuable advice during the course of this study. I am indebted to Prof. S. Uchida, Ocean Research Institute, University of Tokyo, for supplying the eels.     

Monoaminhaltige Strukturen im Zentralnervensystem der Trichoptera (Insecta) Teil II

Zusammenfassung Mit Hilfe der fluoreszenzmikroskopischen Methode nach Falck und Hillarp wurden die monoaminhaltigen Strukturen im Zentralnervensystem einiger Trichopteren untersucht (vgl. Klemm, 1968). Im Protocerebrum können vier Gruppen von katecholamin-haltigen Zellkörpern unterschieden werden. Eine weitere unregelmäßig darstellbare Gruppe von globulösen Perikarya liegt im Lobus opticus. Fluoreszierende Varikositäten durchsetzen locker das Cerebralganglion und sind in folgenden Neuropilstrukturen konzentriert: Medulla, Lobula, Corpus centrale, Nodulus, Corpus ventrale, - und -Lobus und Lobus communis. Letzterer verbindet den - und den -Lobus mit dem nicht fluoreszierenden Pedunculus. In der Lamina, in der accessorischen Medulla, in der Pons cerebralis, im Tuberculum opticum, im Tractus olfactorio-globularis, im Pedunculus, im Stielglomerulus und in den Globulizellen ließen sich keine Katecholamine nachweisen. Zwischen dem monoaminhaltigen Lobus communis und dem nicht fluoreszierenden Pedunculus besteht eine scharfe Grenze. Fluoreszierende variköse Fasern und einzelne fluoreszierende Perikarya befinden sich im Deuto- und Tritocerebrum. Zwei fluoreszierende Bahnen können im Cerebralganglion unterschieden werden: 1. Stratum caudale, 2. Tractus ventralis. Letzterer beginnt mit seiner Pars anterior und posterior im dorsalen fluoreszierenden Neuropil des Protocerebrum. Die beiden Teile laufen frontal und caudal um den Zentralkörper herum und vereinigen sich unterhalb des Zentralkörpers in der Pars communis. Von hier aus zieht der paarige Tractus ventralis bis in das Tritocerebrum. Die Frage einer Homologisierung dieser Bahnen wird diskutiert.Ähnlich wie im Ganglion suboesophageale sind in den drei thorakalen und fünf separaten abdominalen Ganglien zwei Paar fluoreszierender Zellkörper vorhanden, in denen Dopamin mikrospektrofluorimetrisch festgestellt werden konnte. Ein Paar dieser Zellen liegt caudoventral, das andere dorso-median bis dorso-caudal. Ihre Zellfortsätze und Abzweigungen werden beschrieben. In einzelnen Fällen konnte noch ein zweites dorsales Paar fluoreszierender Perikarya sichtbar gemacht werden. Das letzte Ganglion der Bauchkette setzt sich aus zwei bis drei Gangliomeren zusammen. Die Anzahl ihrer fluoreszierenden Perikarya ist reduziert. Das Neuropil der Ganglien im Bauchmark ist von monoaminhaltigen Fasern durchsetzt, wobei sich in der dorsalen Hälfte mehr fluoreszierendes Neuropil befindet als in der ventralen. Lateral in den Ganglien sind die monoaminhaltigen varikösen Fasern vorwiegend dorsoventrad angeordnet. Im medianen Teil laufen sie in Längsrichtung, verzweigen sich und setzen sich in die Konnektive fort und verbinden die katecholaminhaltigen Neuropilbereiche der einzelnen Ganglien miteinander.

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Monoamine-containing structures in the central nervous system in Trichoptera (Insecta). Part II

Summary The distribution of monoamine-containing structures in the central nervous system of Trichoptera has been investigated with the histochemical fluorescence method of Falck and Hillarp (cf. also Klemm, 1968). In the protocerebrum, four groups of catecholamine containing perikarya can be distinguished. Another group of irregularly appearing cell bodies is situated in the optical lobe. Fluorescent varicosities occur scattered throughout the cerebral ganglion, being concentrated in the following neuropile areas: In the medulla, the lobula, the corpus centrale, the noduli, the corpora ventralia, and the - and -lobes, as well as in the lobus communis where the - and -lobes join the non-fluorescent pedunculus. In the lamina, the accessory medulla, the pons cerebralis, the tuberculum opticum, the tractus olfactorio-globularis, the pedunculus including its glomerular region, and the globuli-cells, no catecholamines are detectable. There is a sharp borderline between the monoamine-containing lobus communis and the non-fluorescent pedunculus. Fluorescent varicose fibres and single fluorescent perikarya occur in the Deuto- and Tritocerebrum. Two fluorescent tracts can be distinguished in the cerebral ganglion: 1. Stratum caudale. 2. Tractus ventralis. The pars anterior and pars posterior of the tractus ventralis begin in the dorsal fluorescent neuropile of the cerebral ganglion and join underneath the central body into their pars communis. From this, it can be traced as tractus ventralis into the tritocerebrum. The possible homologies of these tracts are discussed. Similar to the ganglion suboesophageale, the three thoracal and the five separate abdominal ganglia contain two paires of fluorescent cell bodies. Microspectrofluorometrically a content of dopamine in these cells could be established. One pair lies caudoventrally. The two other cell bodies are situated dorso-medially to dorso-caudally; their position varies notably, especially in the abdominal ganglia. The cell process and its arborisations are described. Occasionally a second dorsal cellpair could be observed. The last ganglion of the abdominal chain is composed of at least two gangliomeres with a reduced number of fluorescent perikarya. The neuropile of the thoracal- and abdominal ganglia is penetrated by monoamine-containing fibres, with a predominance in the dorsal half. In the ganglia, the fluorescent varicose fibres are mainly oriented dorso-ventrally in the lateral part and longitudinally in the medial part, where they branch and continue into the connectives. In this way, the catecholamine-containing neuropiles of adjacent ganglia are connected to each other.

Die vorliegende Arbeit wurde unterstützt durch The Swedish Medical Research Council (No. B70-14X-56-06 und B70-14X-05).     

4-[18F]Fluoro-L-m-Tyrosine: An L-3,4-Dihydroxyphenylalanine Analog for Probing Presynaptic Dopaminergic Function with Positron Emission Tomography

4-[18F]Fluoro-L-m-tyrosine (FMT), a biochemical probe of striatal dopaminergic function, has been synthesized as an L-3,4-dihydroxyphenylalanine analog for positron emission tomography. Biochemical characterization of this compound in the rat 30 min after intrajugular administration indicated that in the brain, selective decarboxylation occurred in the striatum, with the formation of 4-fluoro-3-hydroxyphenylethylamine and its metabolites. Positron emission tomography analysis of brain tissue in monkeys (Macaca nemestrina) after intravenous injection of FMT revealed a true time-dependent, specific accumulation of radioactivity in striatum, with a striatum/cerebellum (nonspecific) ratio of 4 at 180 min. Peripheral metabolism accounted for less than 40% of the total radioactivity in arterial blood samples after 120 min. The amino acid remained as the major component throughout the period of investigation (n = 3; 5 min, 95%; 10 min, 85%; 30 min, 67%; 60 min, 62%; 120 min, 60%), with a plasma clearance t 1/2 of 112 min. 3-O-Methylated metabolites were not observed. The substrate specificity of FMT, coupled with its limited in vivo peripheral metabolism, makes it a useful, new biochemical probe for in vivo, noninvasive evaluation of central dopaminergic mechanisms.     

Molecular insights into human monoamine oxidase (MAO) inhibition by 1,4-naphthoquinone: evidences for menadione (vitamin K3) acting as a competitive and reversible inhibitor of MAO

Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic and exogenous amines and its inhibitors have therapeutic value for several conditions including affective disorders, stroke, neurodegenerative diseases and aging. The discovery of 2,3,6-trimethyl-1,4-naphthoquinone (TMN) as a nonselective and reversible inhibitor of MAO, has suggested 1,4-naphthoquinone (1,4-NQ) as a potential scaffold for designing new MAO inhibitors. Combining molecular modeling tools and biochemical assays we evaluate the kinetic and molecular details of the inhibition of human MAO by 1,4-NQ, comparing it with TMN and menadione. Menadione (2-methyl-1,4-naphthoquinone) is a multitarget drug that acts as a precursor of vitamin K and an inducer of mitochondrial permeability transition. Herein we show that MAO-B was inhibited competitively by 1,4-NQ (K_i = 1.4 μM) whereas MAO-A was inhibited by non-competitive mechanism (K_i = 7.7 μM). Contrasting with TMN and 1,4-NQ, menadione exhibited a 60-fold selectivity for MAO-B (K_i = 0.4 μM) in comparison with MAO-A (K_i = 26 μM), which makes it as selective as rasagiline. Fluorescence and molecular modeling data indicated that these inhibitors interact with the flavin moiety at the active site of the enzyme. Additionally, docking studies suggest the phenyl side groups of Tyr407 and Tyr444 (for MAO-A) or Tyr398 and Tyr435 (for MAO-B) play an important role in the interaction of the enzyme with 1,4-NQ scaffold through forces of dispersion as verified for menadione, TMN and 1,4-NQ. Taken together, our findings reveal the molecular details of MAO inhibition by 1,4-NQ scaffold and show for the first time that menadione acts as a competitive and reversible inhibitor of human MAO.     

Elevated oxidase and esterase levels associated with permethrin tolerance in Anopheles gambiae from Kenyan villages using permethrin-impregnated nets

The permethrin tolerance (PT) of a population of the mosquito Anopheles gambiae (Diptera: Culicidae) increased following the introduction of permethrin-impregnated nets for malaria control in certain villages near Kisumu, western Kenya. Using a biochemical test that indirectly measures oxidases associated with permethrin resistance, we found that this population had higher oxidase levels than a comparison population from villages without impregnated nets. Mosquitoes from a colony of An. gambiae selected for PT, the RSP (reduced susceptibility to permethrin) strain, were exposed to permethrin with or without the oxidase inhibitor piperonyl butoxide (PB). Significantly higher mortality rates occurred when permethrin was synergized by PB, presumably by suppression of oxidases responsible for PT. An unselected (UNS) colony of An. gambiae that was more susceptible than RSP in a permethrin-susceptibility bioassay (i.e. LT50 22 min for UNS, vs. 42min for RSP) was compared with the RSP colony for levels of oxidases and esterases. The levels of both enzymes were very significantly higher in the RSP strain (P<0.0001). We speculate that use of impregnated nets selected for higher oxidase and esterase levels in An. gambiae to metabolize permethrin acquired from the nets. Both oxidase and esterase mechanisms could confer cross-resistance to other pyrethroids.