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Ecdysone-mediated stimulation of DOPA decarboxylase activity and its relationship to ovarian development in Aedes aegypti.

Very little dopa decarboxylase activity is detectable in adult female mosquitoes Aedes aegypti which have not been allowed to engorge blood. However, when such females are injected with the molting hormone beta-ecdysone a marked stimulation of this enzyme's activity is observable. No stimulation is observed in males similarly injected, nor in females injected with cholesterol or a juvenile hormone mimic. In addition, ecdysone injection initiates ovarian development in these anautogenous non-blood-fed mosquitoes. The extent of stimulation in both cases is dependent upon the amount of beta-ecdysone administered. These results suggested that ecdysone may play a role in ovarian development in Aedes and led us to hypothesize that a normal blood meal may trigger the synthesis, activation, or release of this hormone endogenously. Using the radioimmune assay for ecdysone developed by Borst and O'Connor (Science [Wash. D. C.] 178:4-18.), we found that the titer of an antigenic-positive material, presumably ecdysone or a closely related analogue, substantially increased 24 h after blood feeding, thereby supporting our postulation.

Aedes↗

Sequence and expression of the Cc gene, a member of the dopa decarboxylase gene cluster of Drosophila: possible translational regulation.

A transcript has been localized proximal to the dopa decarboxylase (Ddc) gene within a cluster of genes involved in cuticle formation and catecholamine metabolism in Drosophila. This gene, which has been identified as I(2)37Cc, maps 2.0kb from the 5' end of the Ddc gene and is transcribed in the same direction as Ddc. We describe a new deficiency which in conjunction with previous deficiencies localizes the I(2)37Cb and I(2)37Cc loci to the cytogenetic interval 5' to Ddc. We present the sequence of the Cc gene and corresponding cDNA. The Cc message contains several open reading frames 5' to the large open reading frame responsible for the lethal complementation group, suggesting that expression of Cc function may be regulated translationally. The Cc transcript is expressed in early embryos, late embryos, late third instar larvae and adults. We discuss the implications of these findings with respect to the gene organization in the region.

Animals↗

L-dopa decarboxylase in Ceratitis capitata white puparia and human: a comparative study.

1. L-DOPA decarboxylase (DDC) from Ceratitis capitata and from human kidney have been purified by the same methodology. 2. Both enzymes show mol. wts of 100,000, consisting of two identical mol. wt subunits and solely decarboxylate L-DOPA. 3. In the presence of 5-hydroxytryptophan (5-HTP) only the DDC activity from human kidney is remarkably reduced. 4. Addition of exogenous coenzyme is essential only for human DDC activity. 5. Polyclonal antibodies, raised against DDC purified from insects or humans, cross-react with both antigens.

Animals↗

Observations on the substrate specificity of DOPA decarboxylase from ox adrenal medulla, human phaeochromocytoma and human argentaffinoma.

The substrate specificity of the dopa decarboxylases of ox adrenal medulla, human phaeochromocytoma and human argentaffinoma have been studied. The enzymes from all three tissues decarboxylated dopa, metatyrosine, orthotyrosine and 5-hydroxytryptophan. Dopa was decarboxylated most rapidly and 5-hydroxytryptophan least rapidly by the enzyme from all three tissues. Competition experiments indicate that all four substrates are decarboxylated by the one enzyme. Attempts to demonstrate decarboxylation of [C(14)]-tyrosine, [C(14)]-tryptophan or [C(14)]-histidine by these enzyme preparations were unsuccessful.

5-Hydroxytryptophan↗

Modulation of novel-length DOPA decarboxylase transcripts by 20-OH-ecdysone in a Drosophila melanogaster Kc cell subline.

The induction of DOPA decarboxylase (DDC) activity by 20-OH-ecdysone (20-OHE) in a subline of Drosophila melanogaster Kc cells was investigated. Cells cultured in the continuous presence of the steroid hormone exhibited a 96-h temporal lag prior to a peak of DDC enzyme activity while arrested in the G2 phase of the cell cycle. The concentration of Ddc RNA increased sixfold between 72 and 96 h after initial exposure to hormone. Similarly, this increase was correlated temporally with a 26-fold increase in DDC enzyme activity. The Kc Ddc primary transcript, processing intermediate, and mature mRNA all were approximately 500 nucleotides longer than the corresponding transcripts observed for newly eclosed adult D. melanogaster. In vitro translation of poly(A)+ RNA from Kc cells resulted in an immunoprecipitable polypeptide which exhibited similar mobility on sodium dodecyl sulfate gels to that of DDC synthesized in vitro by larval epidermal poly(A)+ RNA.

Animals↗

Effect of L-dopa on plasma renin activity with and without inhibition of extracerebral dopa decarboxylase in dogs.

The aim of the present study was to investigate the possibility that catecholaminergic pathways within the central nervous system play a role in the control of renin secretion. Plasma renin activity (PRA) was measured after intravenous administration of L-dopa with and without prior inhibition of extracerebral dopa decarboxylase by carbidopa (MK-486) in pentobarbital-anesthetized dogs in which changes in renal perfusion pressure were minimized by means of a suprarenal aortic clamp. When L-dopa (20 mg/kg ) was administered intravenously without carbidopa. PRA and blood pressure increased. In contrast, administration of L-dopa (20 mg/kg i.v.) after blockade of extracerebral, but not cerebral dopa decarboxylase by intravenous carbidopa (20 mg/kg), produced significant decrease in both PRA and blood pressure. Larger doses of l-dopa (30-50) mg/kg i.v.) also lowered both PRA and blood presure in three carbidopa-treated animals, whereas a smaller dose of L-dopa (10 mg/kg i.v.) significantly lowered blood pressure but not PRA. In dogs with both kidneys acutely denervated, L-dopa (20 mg/kg i.v.) with carbidopa lowered arterial pressure but did not consistently alter PRA. These data suggest that catecholamines formed within the central nervous system can act to lower renin secretion as well as blood pressure.

Animals↗

Construction of an unstable Ring-X chromosome bearing the autosomal dopa decarboxylase gene in Drosophila melanogaster and analysis of Ddc mosaics.

An unstable Ring-X chromosome, Ddc+ -Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)wvC and a Rod-X chromosome which contained a P-element mediated Ddc+ insert. The resulting Ddc+ -Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc+ -Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc+ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.

Animals↗

Isolation and characterization of a dopa decarboxylase cDNA and the induction of its expression by an insect cytokine, growth-blocking peptide in Pseudaletia separata.

Parasitization by the wasp, Cotesia kariyai, elevates the concentration of an insect cytokine, growth-blocking peptide (GBP), in hemolymph of last instar Pseudaletis separata larvae. The increase of epidermal and hemolymph dopamine level is associated with the GBP increase. Both GBP and dopamine disturb host development and metamorphosis (Hayakawa, 1995). Dopa decarboxylase (DDC) converts Dopa to dopamine, and its cDNA was isolated from P. separata, and the deduced amino acid sequence showed that it was highly homologous to other lepidopteran DDCs, showing 96, 90 and 86% identity with those of Mamestra brassicae, Bombyx mori, and Manduca sexta, respectively. A 3.2 kb DDC mRNA transcript was constitutively expressed at low levels in the epidermis, brain-nerve cord and hemocytes, and the expression was enhanced by injection of GBP in these tissues. Detailed characterization of the DDC mRNA expression in the epidermis showed that its expression reached a plateau 3 hr after the injection. DDC activity and DDC protein (55 kDa) level mirrored the mRNA expression. Immunocytochemistry with anti-DDC antibody confirmed that the enhanced DDC expression was localized in the epidermal cells. Dopamine concentration in the epidermis gradually increased and reached maximum 6 hr after the injection. When the epidermis of Day 1 last instar larvae was cultured in vitro in the presence of GBP, DDC mRNA increased, indicating that GBP acted on the epidermal cells directly to induce expression of the DDC gene.

Amino Acid Sequence↗

Phylogenetic utility of the nuclear gene dopa decarboxylase in noctuoid moths (Insecta: Lepidoptera: noctuoidea).

Phylogenetic utility for the nuclear gene encoding dopa decarboxylase (DDC), little used in systematics, was recently demonstrated within the noctuid moth subfamily Heliothinae. Here we extend the test of the utility of a 709-bp DDC fragment to deeper levels, analyzing 49 species representing major groups across the superfamily Noctuoidea. Parsimony, distance, and maximum-likelihood analyses recover all or nearly all of a set of "test clades" supported by clear morphological synapomorphies, spanning a wide range of taxonomic levels. DDC also upholds a recent proposal that the Noctuidae are paraphyletic. Nt3 contributes a majority of the signal and recovers the basal split between Notodontidae and all other noctuoids, despite a plateau of nt3 divergence at this level. However, nonsynonymous changes also support groups at all levels, and in contrast to nt3, amino acid divergence shows no plateau. The utility of DDC promises to extend back to the early Tertiary and Cretaceous, a time span for which few suitable genes have been identified.

Animals↗

The genetic and molecular organization of the Dopa decarboxylase gene cluster of Drosophila melanogaster.

We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.

Alleles↗

Dopa decarboxylase genotypes may influence age at onset of schizophrenia.

Several lines of evidence implicate dopa decarboxylase (DDC) with schizophrenia. By analysis of two putative functional DDC variants in 173 schizophrenic patients and 204 controls we tested the hypotheses that DDC is involved in: (1) predisposition to schizophrenia; and (2) modulation of age at disease onset. No association was observed with schizophrenia as a whole, whereas an association between DDC genotypes and age at disease onset was suggested in males (P = 0.03). This association was most pronounced in relation to genotypes of haplotypes comprising both variants, suggesting an additive model where one variant mediates early and the other late onset. Accordingly, the haplotype-based genotypes could be assigned into three groups by their possible relative effect on age at onset: an "early", "neutral" and "late" group. Dividing the male schizophrenics into four groups with increasing age at onset, the "early" genotypes were seen to decrease in frequency from 51.5% to 16.7% while the "late" genotypes increased from 12.1% to 33.3% (P = 0.02). The difference in mean age at onset between male patients with "early" genotypes vs patients with "late" genotypes was close to 5 years (95% CI: 0.7-8.8). Thus, DDC may possibly act as a modulator of age at onset in male schizophrenics.

Adult↗

A controlled study of a dopa decarboxylase inhibitor (benserazide) in the treatment of schizophrenic patients.

In a double-blind controlled study lasting 6 weeks, 32 schizophrenic patients were randomly assigned to one of two treatments: chlorpromazine or benserazide, a dopa decarboxylase inhibitor. Results on each measure were subject to multifactorial analysis of covariance. Benserazide did not appear to be as effective an antipsychotic medicaiton as chlorpromazine. In fact, chlorpromazine was significantly better on measures of tension, excitement, hallucinatory behavior, thinking disturbance, social interest, and drop-out rate. At the relatively low dosages used in this study, it would seem that benserazide had little effect on the cerebral dopa decarboxylase, and it would be worthwhile trying higher dosages.

Adult↗

Temporal and spatial expression of the yellow gene in correlation with cuticle formation and dopa decarboxylase activity in Drosophila development.

The yellow (y) gene of Drosophila is required for the formation of black melanin and its deposition in the cuticle. We have studied by immunohistochemical methods the temporal and spatial distribution of the protein product of the y gene during embryonic and pupal development and have correlated its expression with events of cuticle synthesis by the epidermal cells and with cuticle sclerotization. Except for expression in early embryos, the y protein is only found in the epidermal cells and may be secreted into the cuticle as it is being deposited. The amount of y protein in various regions of the embryo and pupa correlates directly with the intensity of melanization over any section of the epidermis. Expression of the y gene begins in the epidermal cells at 48 hr after pupariation and is well correlated with the beginning deposition of the adult cuticle. At this stage the adult cuticle is unsclerotized and unpigmented and dopa decarboxylase levels, a key enzyme in catecholamine metabolism which provides the crosslinking agents as well as the precursors for melanin, is low. As a separate event 26 hr after the onset of y gene expression, the first melanin deposition occurs in the head bristles and pigmentation continues in an anterior to posterior progression until eclosion. This melanization wave is correlated with elevated dopa decarboxylase activity. Crosslinking of the adult cuticle also occurs in a similar anterior to posterior progression at about the same time. We have shown by imaginal disc transplantation that timing of cuticle sclerotization depends on the position of the tissue along the anterior-posterior axis and that it is not an inherent feature of the discs themselves. We suggest that actual melanization and sclerotization of the cuticle by crosslinking are initiated at this time in pupal development by the availability of the catecholamine substrates which diffuse into the cuticle. Intensity of melanization and position of melanin pigment is determined by the presence or absence of the y protein in the cuticle, thus converting the y protein prepattern into the melanization pattern.

Animals↗

Analysis of the transcription unit adjacent to the 3'-end of the dopa decarboxylase gene in Drosophila melanogaster.

Using strand specific RNA probes, we have ascertained that the transcription unit immediately downstream of the dopa decarboxylase (DDC) gene in Drosophila, is oriented in the opposite direction to DDC. The 3'-termini of these two transcription units lie within 550 bp of one another, and the mature transcripts actually may overlap. We show here that the inclusion of part of the 3'-flanking transcript on a genomic DNA clone originally assumed to include just DDC, accounts for most of the hybridization seen in our previous Northern analysis of very early embryonic RNA. Thus, while DDC-specific transcripts are detectable in the early embryonic stages, they are much less prevalent than we have previously reported.

Animals↗

Striatal L-dopa decarboxylase activity in Parkinson's disease in vivo: implications for the regulation of dopamine synthesis.

L-DOPA is a large neutral amino acid subject to transport out of, as well as into, brain tissue. Competition between dopamine synthesis and L-DOPA egress from striatum must favor L-DOPA egress if decarboxylation declines relatively more than transport in Parkinson's disease. To test this hypothesis, we injected patients with Parkinson's disease with a radiolabeled analogue of L-DOPA and recorded regional brain radioactivity as a function of time by means of positron emission tomography. We simultaneously estimated the activity of the decarboxylating enzyme and the amino acid transport. In the striatum of patients, we found the L-DOPA decarboxylase activity to be reduced in the head of the caudate nucleus and the putamen. However, the rate of egress of the DOPA analogue was unaffected by the disease and thus inhibited dopamine synthesis more than predicted in the absence of L-DOPA egress.

Corpus Striatum↗

A neuron-specific enhancer of the Drosophila dopa decarboxylase gene.

At least two cis-regulatory elements are necessary for correct neuron-specific expression of the Drosophila melanogaster dopa decarboxylase gene, Ddc. In addition to a previously described proximal element located approximately 60 bp upstream of the mRNA start site, we have now characterized a distal approximately 600-bp DNA fragment, extending from -1019 to -1623 bp, which possesses enhancer-like properties and is essential for normal neuron-specific expression. Immunofluorescent labeling of neurons expressing deleted Ddc genes indicates that this region contains both general neuronal regulatory elements and cell-specific elements that selectively affect Ddc expression in either dopaminergic or serotonergic neurons. These selective effects can be correlated with the removal of sequence elements that are protected from DNase digestion by factors present in embryonic nuclear extracts. Several of these elements are also homologous to sequences located upstream of the evolutionarily diverged Ddc gene of Drosophila virilis. These results suggest that the neuron-specific expression of Ddc results from the combined action of several factors binding within this distal enhancer region.

Animals↗

In vivo regulation of DOPA decarboxylase by dopamine receptors in rat brain.

To test the hypothesis that dopamine (DA) receptors influence cerebral DOPA-decarboxylase (DDC) activity in vivo, we used HPLC to measure the kinetics of the cerebral uptake and metabolism of [3H]DOPA in carbidopa-treated rats, and in rats also treated acutely with a DA receptor antagonist (flupenthixol, 2 mg/kg, intraperitoneally) or a DA receptor agonist (apomorphine, 200 microg/g, subcutaneously). The unidirectional blood-brain clearance of [3H]DOPA (K1DOPA, 0.030 mL g(-1) min(-1)) increased by 50% after flupenthixol. The magnitudes of the relative DDC activity (k3DOPA) in striatum (0.20 min(-1)), olfactory tubercle (0.11 min(-1)), and hypothalamus (0.15 min(-1)) of carbidopa-treated rats were doubled with flupenthixol, but cortical DDC activity was unaffected (0.02 min(-1)). Apomorphine reduced the magnitude of k3DOPA in striatum by 20%. The rate constant for catabolism of [3H]DA formed in brain (k7', monoamine oxidase [MAO] activity), which ranged from 0.025 min(-1) in striatum to 0.08 min(-1) in hypothalamus of carbidopa-treated rats, globally increased 2- to 4-fold after flupenthixol, and decreased to 0.003 min(-1) in striatum after apomorphine. These in vivo results confirm the claim that acute blockade of DA receptors with flupenthixol stimulates the synthesis of [3H]DA from [3H]DOPA, and that this [3H]DA is subject to accelerated catabolism. Conversely, activation of the DA receptors with apomorphine inhibits DDC activity and DA catabolism.

Animals↗

Aromatic amino acid methyl ester analogs form quinonoidal species with Dopa decarboxylase.

This study reports for the first time that binding of aromatic methyl ester analogs to Dopa decarboxylase in the native and inactive nicked forms causes the appearance of a dead-end quinonoidal species absorbing at 500 nm, in addition to an external aldimine absorbing at 398 nm. The equilibrium mixture of these species varies depending on both the analog structure and the enzyme form. The above mentioned intermediates are also characterized with respect to their CD properties and the equilibria for their formation are determined as a function of pH. The results have provided evidence that the establishment of proper contacts between the active site and hydroxyl groups of the ligand are indispensable in order to limit unwanted side reactions.

Amino Acids↗