A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus.
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Aspergillus fumigatus, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for "albino 1"), arp1 (for "aspergillus reddish-pink 1"), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for "aspergillus brown 1") encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans. The function of ayg1 (for "aspergillus yellowish-green 1") remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus. The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus. However, the presence of abr1, abr2, and ayg1 in addition to alb1, arp1, and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway. (+info)
A comparison of 11C-labeled L-DOPA and L-fluorodopa as positron emission tomography tracers for the presynaptic dopaminergic system.
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11C-labeled 3,4-Dihydroxy-phenyl-L-alanine (L-DOPA) and L-fluorodopa were used as tracers for the functional state of the presynaptic dopamine system in anesthetized monkeys with positron emission tomography. The radiotracer disposition in brain tissue and plasma were studied and effects induced by pharmacologic challenges were evaluated. 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH4) increased the striatal influx rate constant, e.g., striatal K(i) for L-[beta-11C]DOPA, but it induced no effect on the K(i)-value using L-[beta-11C]-6-fluorodopa. Studies of radiolabeled tracer and metabolites in plasma showed substantial differences between the two tracers. At baseline conditions, 60% unchanged L-[beta-11C]DOPA was detected in plasma 50 minutes after tracer injection and the 3-O-methylated fraction accounted for 25% of total radioactivity. For L-[beta-11C]-6-fluorodopa, the relation was inverse; about 25% unchanged tracer and 60% 3-O-methyl metabolite were present in plasma after 50 minutes. A site-specific 11C-labeling in the carboxylic position in the molecules revealed a significant specific retention of radioactivity in striatum with L-[car-boxy-11C]-6-fluorodopa but not with L-[carboxy-11C]DOPA. The 3-O-methyl metabolite of L-DOPA is known to pass the blood-brain barrier and may interfere with the calculation of the K(i)value using a brain reference region. Thus, extensive 3-O-methylation in circulation of the fluorinated analog could obscure the detectability of potential functional change in striatal K(i) of the tracer when using a reference tissue model for calculation. (+info)
Visualization of dioxygen bound to copper during enzyme catalysis.
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X-ray crystal structures of three species related to the oxidative half of the reaction of the copper-containing quinoprotein amine oxidase from Escherichia coli have been determined. Crystals were freeze-trapped either anaerobically or aerobically after exposure to substrate, and structures were determined to resolutions between 2.1 and 2.4 angstroms. The oxidation state of the quinone cofactor was investigated by single-crystal spectrophotometry. The structures reveal the site of bound dioxygen and the proton transfer pathways involved in oxygen reduction. The quinone cofactor is regenerated from the iminoquinone intermediate by hydrolysis involving Asp383, the catalytic base in the reductive half-reaction. Product aldehyde inhibits the hydrolysis, making release of product the rate-determining step of the reaction in the crystal. (+info)
Modulation of fictive feeding by dopamine and serotonin in aplysia.
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The buccal ganglia of Aplysia contain a central pattern generator (CPG) that mediates rhythmic movements of the buccal apparatus during feeding. Activity in this CPG is believed to be regulated, in part, by extrinsic serotonergic inputs and by an intrinsic and extrinsic system of putative dopaminergic cells. The present study investigated the roles of dopamine (DA) and serotonin (5-HT) in regulating feeding movements of the buccal apparatus and properties of the underlying neural circuitry. Perfusing a semi-intact head preparation with DA (50 microM) or the metabolic precursor of catecholamines (L-3-4-dihydroxyphenylalanine, DOPA, 250 microM) induced feeding-like movements of the jaws and radula/odontophore. These DA-induced movements were similar to bites in intact animals. Perfusing with 5-HT (5 microM) also induced feeding-like movements, but the 5-HT-induced movements were similar to swallows. In preparations of isolated buccal ganglia, buccal motor programs (BMPs) that represented at least two different aspects of fictive feeding (i.e., ingestion and rejection) could be recorded. Bath application of DA (50 microM) increased the frequency of BMPs, in part, by increasing the number of ingestion-like BMPs. Bath application of 5-HT (5 microM) did not significantly increase the frequency of BMPs nor did it significantly increase the proportion of ingestion-like BMPs being expressed. Many of the cells and synaptic connections within the CPG appeared to be modulated by DA or 5-HT. For example, bath application of DA decreased the excitability of cells B4/5 and B34, which in turn may have contributed to the DA-induced increase in ingestion-like BMPs. In summary, bite-like movements were induced by DA in the semi-intact preparation, and neural correlates of these DA-induced effects were manifest as an increase in ingestion-like BMPs in the isolated ganglia. Swallow-like movements were induced by 5-HT in the semi-intact preparation. Neural correlates of these 5-HT-induced effects were not evident in isolated buccal ganglia, however. (+info)
Physiological importance of quinoenzymes and the O-quinone family of cofactors.
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O-quinone cofactors derived from tyrosine and tryptophan are involved in novel biological reactions that range from oxidative deaminations to free-radical redox reactions. The formation of each of these cofactors appears to involve post-translational modifications of either tyrosine or tryptophan residues. The modifications result in cofactors, such as topaquinone (TPQ), tryptophan tryptophylquinone (TTQ), lysine tyrosylquinone (LTQ) or the copper-complexed cysteinyl-tyrosyl radical from metal-catalyzed reactions. Pyrroloquinoline quinone (PQQ) appears to be formed from the annulation of peptidyl glutamic acid and tyrosine residues stemming from their modification as components of a precursor peptide substrate. PQQ, a primary focus of this review, has invoked considerable interest because of its presence in foods, antioxidant properties and role as a growth-promoting factor. Although no enzymes in animals have been identified that exclusively utilize PQQ, oral supplementation of PQQ in nanomolar amounts increases the responsiveness of B- and T-cells to mitogens and improves neurologic function and reproductive outcome in rodents. Regarding TPQ and LTQ, a case may be made that the formation of TPQ and LTQ is also influenced by nutritional status, specifically dietary copper. For at least one of the amine oxidases, lysyl oxidase, enzymatic activity correlates directly with copper intake. TPQ and LTQ are generated following the incorporation of copper by a process that involves the two-step oxidation of a specified tyrosyl residue to first peptidyl dopa and then peptidyl topaquinone to generate active enzymes, generally classed as "quinoenzymes." Limited attention is also paid to TTQ and the copper-complexed cysteinyl-tyrosyl radical, cofactors important to fungal and bacterial redox processes. (+info)
A study of hypothalmic neurosecretory cells of bullfrogs in vitro.
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Neurosecretory cells of preoptic nuclei of bullfrogs were studied in isolated hypothalamo-hypophysial preparations under constant perfusion with oxygenated Ringer solution at 15-17 degress C. Antidromic potentials were recorded following stimuli applied to the posterior lobe of the pituitary or the stalk. 2. Intracellularly and extracellularly recorded potentials resembled those obtained in vivo from neurosecretory cells of the mammalian hypothalamus. They were unique in that the antidromic potential had a long duration (10-20 msec) and a distinct notch on the rising phase (between A and B spikes). The conduction velocity of the stalk fibres in vitro at this temperature was 0-1--0-2 m/sec. 3. When two successive stimuli were given to the posterior lobe or to the stalk separated by intervals of between 30 and 65 msec, the test (second) response showed a longer delay of the B spike. This delay between the A and B components was as long as 10 msec. Further shortening of stimulus intervals produced block of B spikes in the test response. A complete separation of A and B spikes occur spontaneously in a few instances. 4. Evidence indicated that inhibitory recurrent axon collaterals play a role in the control of bullfrog neurosecretory cells. Antidromic potentials were inhibited by a 'conditioning' stimulus for as long as 300-400 msec, even when the stimulus did not evoke an antidromic potential. 5. It was found that in addition to the inhibitory interaction there is a facilitatory recurrent axon collateral system which operates within the nuclei. The evidence for this is: (1) stimulation of the posterior lobe, with single subthreshold pulses evoked an action potential if preceded by another stimulus of subthreshold or just threshold intensity. The durations of such facilitatory effects were found to be 20--400 msec; (2) a single pulse given to the posterior lobe did occasionally evoke two spikes from neurosecretory cells; the second spike which occurred 15-30 msec after the first had the characteristics of a trans-synaptically produced potential; (3) gradual changes in the intensity of stimuli applied to the neural lobe produced a sudden shift in latencies ranging between 15 and 30 msec. The potentials having long latency also showed characteristics of those transsynaptically excited. In addition, an increase in excitability of neurosecretory cells by antidromic stimulation was confirmed by using orthodromically induced action potentials in in vivo studies. Possible functional significance of inhibitory and excitatory recurrent collateral system in neurosecretory cells was discussed. 6. Two to threefold increase in NaCl concentration of a perfusate slightly increased the latency and refractory period of antidromic potential but not the shape of the potential. Norepinephrine added to a perfusate (1 mug/ml). augmented the separation of A and B spikes of the antidromic potential. Acetylcholine at a concentration of 1 mug/ml. did not have an appreciable effect on the antidromic potential. (+info)
The oxidation and reduction reactions of bovine serum amine oxidase. A kinetic study.
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The presteady-state and steady-state kinetics of bovine serum amine oxidase (BSAO) were analyzed by stopped-flow transient spectroscopy. A simplified model of the catalytic cycle was found to describe the experimental data and the rate constants of the individual steps were used to calculate Michaelis parameters that agree with the direct determinations. In spite of many studies on selected reactions from the catalytic cycle, this is amongst the first efforts to provide a comprehensive kinetic description of the reactions of BSAO, whose results can be compared with the steady-state parameters. The reoxidation reaction by dioxygen is more complex than previously thought, in agreement with a recent report [Su, Q. & Klinman, J.P. (1998) Biochemistry 37, 12513-12525], and occurs in at least two steps whose rate constants, previously undetermined, have been measured. The reaction of the oxidized enzyme with the amine substrate is poorly determined in this type of experiment, thus irreversible combination with aromatic hydrazine inhibitors was used as a model system, demonstrating that the mechanism and rate constants of their reaction is fully compatible with an accurate description of the catalytic cycle with the physiological substrate. These results constitute a simplified, yet complete and consistent, description of the catalytic cycle and offer an interesting comparison with those obtained on plant amine oxidases; two steps of the catalytic cycle are significantly slower in BSAO than in pea seedling or lentil seedling amine oxidases, namely the reoxidation and the trans-iminative proton abstraction occurring in the enzyme-substrate complex. The former difference is rationalized as being due to the low to zero concentration of the semiquinolamine-radical intermediate, while the latter is less easily interpreted. (+info)
Inhibitory effect of 5-hydroxytryptophane on the induction of persistent estrus by androgen in the rat.
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Effects of monoamines or its precursors on the induction of persistent estrus by testosterone propionate (T.P.) were studied in Wistar strain female rats. Daily treatment with 5-hydroxytryptophane (5-HTP) for the first 10 days of life delayed the occurrence of persistent estrus in rats given T.P. at 4 days of age. The onset of persistent estrus occurred in this group of animals at 67.1+/-2.5 days of age compared with 45.1+/-1.4 days of age for the saline treated control group. The role of hypothalamic monoamines in contributing to the induction of persistent estrus by T.P. treatment of neonatal rats is discussed. (+info)