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PubMed1990

Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone.

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 micrograms juvenile hormone I at HCS. Infusion of 1 microgram/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 microgram/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1-3 micrograms/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.

Animals↗
PubMed1982

Effects of 3,4-dihydroxyphenylpyruvic acid and its triacetylated derivative on DOPA decarboxylase.

Various drugs have been studied in order to improve the bioavailability of L-DOPA. In the present study, the alpha-ketoacid of L-DOPA, 3,4-dihydroxyphenylpyruvic acid (DHPPA) and its fully acetylated derivative, triacetoxyphenylpyruvic acid (TAPPA), were investigated. These substances were shown to be equally strong DOPA decarboxylase inhibitors in vitro. The inhibitory activity was, however, only about 1/2000 of that of carbidopa and 1/10 of that of L-alpha-methyldopa. In vivo studies in the rat showed that when either DHPPA or TAPPA was given orally concomitantly with L-DOPA, only DHPPA, which is a transaminase inhibitor, enhanced the L-DOPA induced rise in the cerebral dopamine concentration. TAPPA had, however, an enhancing effect on the L-DOPA-induced rise in the serum L-DOPA concentration. The weak L-DOPA-sparing effect of TAPPA could be explained by its inhibitory effect on DOPA decarboxylase and the marked L-DOPA-sparing effect of DHPPA by its action on both transamination and decarboxylation.

Animals↗
PubMed1998

Two different cDNAs code for L-DOPA decarboxylase in the white prepuparium and eclosion developmental stages of the insect Ceratitis capitata1.

In insects the DOPA decarboxylase gene (Ddc) is expressed in the epidermis where dopamine derivatives promote cuticle sclerotization and the central nervous system participating in the synthesis of serotonin and dopamine. The temporal course of Ddc enzyme activity during the white prepuparium and eclosion developmental stages primarily reflects this role. Here we report that a cDNA clone for l-DOPA decarboxylase (EC 4.1.1.28) has been isolated from the white puparium stage of the insect Ceratitis capitata, the cDNA sequence has been determined and it corresponds to an mRNA of 1341 base pairs. The comparison of this cDNA sequence with that of the eclosion stage which has been previously isolated in our laboratory revealed that the cDNA from the eclosion stage codes for protein consisting of 431 amino acid (aa) residues whereas that from the white prepuparium stage for protein of 425 aa residues. In addition there is a region of 33 aa residues close to the N-terminal region of the deduced protein in which marked differences in the aa composition are observed. This result was further confirmed by Northern blot analysis in which mRNAs isolated from both developmental stages were only hybridized with their respective polymerase chain reaction synthesized stage specific probes. These results suggest that during development of the insect C. capitata two different mRNAs are produced at the white prepuparium and the eclosion stages, giving rise to two protein isoforms presumably in response to different signals.

Amino Acid Sequence↗
PubMed1987

Purification of L-dopa decarboxylase from rat liver and production of polyclonal and monoclonal antibodies against it.

L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.

Animals↗
PubMed1980

Activities of L-dopa decarboxylase and diamine oxidase (histaminase) in human lung cancers and decarboxylase as a marker for small (oat) cell cancer in cell culture.

The neuroendocrine [amine precursor uptake (decarboxylase)] properties of small (oat) cell lung cancer (SCC) have suggested that this neoplasm may have a separate histogensis from the other major types of human lung tumors. We now report that a key element of this concept, L-dopa decarboxylase activity, is present in surgical and autopsy tissues from all forms of lung cancer. Values are highest in SCC lesions; however, lung adenocarcinoma tissues can have considerable activity, and values overlap those for SCC and fall between values for SCC and large cell and squamous cell carcinoma. The distribution of diamine oxidase activity is identical except that even more overlap occurs between the major tumor types. These data may provide further evidence that SCC and other human lung cancers could share a common origin in the bronchial mucosa. In cell cuture, the distribution of the two enzyme activities is different. The average L-dopa decarboxylase activity is much higher (seven separate culture lines) than in the in vivo specimens, and it completely separates these cell lines from non-SCC lung tumors (four lines). Diamine oxidase is generally low in both SCC cells and non-SCC cells in culture and does not separate the various cell types. L-Dopa decarboxylase activity thus does appear to be a valuabe marker for separating SCC cells from other lung cancer cells in vitro.

Adenocarcinoma↗
PubMed1990

Characterization of DOPA decarboxylase mRNA in rat pheochromocytoma.

Total poly (A+) RNA has been extracted from rat pheochromocytoma and translated in vitro by means of a reticulocyte lysate system. We show that two antisera, prepared against pig kidney DOPA decarboxylase (DDC) or rat pheochromocytoma DDC, immunoprecipitate an in vitro synthetized 50 kDa polypeptide identified as DDC by competition experiments with pure DDC. The proportion of specific mRNA has been calculated and represents 0.05% of total poly A+ mRNA. Its size has been established by electrophoresis in methylmercuric hydroxide containing agarose gel, corresponding to a 2.2 kb length mRNA.

Animals↗
PubMed1997

Mutation of cysteine 111 in Dopa decarboxylase leads to active site perturbation.

Cysteine 111 in Dopa decarboxylase (DDC) has been replaced by alanine or serine by site-directed mutagenesis. Compared to the wild-type enzyme, the resultant C111A and C111S mutant enzymes exhibit Kcat values of about 50% and 15%, respectively, at pH 6.8, while the K(m) values remain relatively unaltered for L-3,4-dihydroxyphenylalanine (L-Dopa) and L-5-hydroxytryptophan (L-5-HTP). While a significant decrease of the 280 nm optically active band present in the wild type is observed in mutant DDCs, their visible co-enzyme absorption and CD spectra are similar to those of the wild type. With respect to the wild type, the Cys-111-->Ala mutant displays a reduced affinity for pyridoxal 5'-phosphate (PLP), slower kinetics of reconstitution to holoenzyme, a decreased ability to anchor the external aldimine formed between D-Dopa and the bound co-enzyme, and a decreased efficiency of energy transfer between tryptophan residue(s) and reduced PLP. Values of pKa and pKb for the groups involved in catalysis were determined for the wild-type and the C111A mutant enzymes. The mutant showed a decrease in both pK values by about 1 pH unit, resulting in a shift of the pH of the maximum velocity from 7.2 (wild-type) to 6.2 (mutant). This change in maximum velocity is mirrored by a similar shift in the spectrophotometrically determined pK value of the 420-->390 nm transition of the external aldimine. These results demonstrate that the sulfhydryl group of Cys-111 is catalytically nonessential and provide strong support for previous suggestion that this residue is located at or near the PLP binding site (Dominici P, Maras B, Mei G, Borri Voltattorni C. 1991. Eur J Biochem 201:393-397). Moreover, our findings provide evidence that Cys-111 has a structural role in PLP binding and suggest that this residue is required for maintenance of proper active-site conformation.

5-Hydroxytryptophan↗
PubMed1986

The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.

Animals↗
PubMed1986

Regulated splicing produces different forms of dopa decarboxylase in the central nervous system and hypoderm of Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) of Drosophila melanogaster is expressed in the hypoderm and the nervous system and promoter elements mediating differential expression in these tissues have been identified (Scholnick et al., 1986). Here we report an additional mode of regulation; the unique primary transcript of the Ddc gene is spliced to form mRNAs in these two tissues which differ by a single internal exon. In vitro mutagenesis and P-element-mediated transformation were employed to manipulate the tissue-specific expression of these RNAs. This approach demonstrated that regulated splicing rather than differential stability causes the tissue-specific expression of these RNAs and allowed the identification of Ddc enzyme isoforms encoded by each mRNA. The Ddc enzyme in the central nervous system differs from the hypodermal Ddc protein by the addition of 33-35 amino acids on the N terminus.

Animals↗
PubMed1984

Mutations in the dopa decarboxylase gene affect learning in Drosophila.

Fruit flies synthesize several monoamine neurotransmitters. Dopa decarboxylase (Ddc) mutations affect synthesis of two of these, dopamine and serotonin. Both transmitters are implicated in vertebrate and invertebrate learning. Therefore, we bred flies of various Ddc genotypes and tested their learning ability in positively and negatively reinforced learning tasks. Mutations in the Ddc gene diminished learning acquisition approximately in proportion to their effect on enzymatic activity. Courtship and mating sequences of the mutants appeared normal, except for one aspect of male courtship that had previously been shown to be experience dependent. In contrast, the effect on behavior patterns that do not involve learning--phototaxis, geotaxis, olfactory acuity, responsiveness to sucrose--was relatively slight under these conditions. Moderate Ddc mutations affected the acquisition of learned responses while leaving memory retention unaltered. This is in contrast to the mutations dunce , rutabaga , and amnesiac , which primarily affect short-term memory.

Animals↗
PubMed1992

Repression and activation of the Drosophila dopa decarboxylase gene in glia.

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

Animals↗
PubMed1993

Tissue-specific alternative splicing of the Drosophila dopa decarboxylase gene is affected by heat shock.

The Drosophila dopa decarboxylase gene, Ddc, is expressed in the hypoderm and in a small number of cells in the central nervous system (CNS). The unique Ddc primary transcript is alternatively spliced in these two tissues. We investigated whether Ddc splicing in the CNS is a general property of the CNS or a unique property of the cells that normally express Ddc by expressing the Ddc primary transcript ubiquitously under the control of an Hsp70 heat shock promoter. Under basal expression conditions, Ddc splicing shows normal tissue specificity, indicating that the regulation of Ddc splicing in the CNS is tissue specific rather than cell specific. Previous studies have shown that severe heat shock blocks mRNA splicing in cultured Drosophila melanogaster cells. Our results show that splicing of the heat shock-inducible Hsp83 transcript is very resistant to heat shock. In contrast, under either mild or severe heat shock, the splicing specificity of the heat shock-induced Ddc primary transcript is affected, leading to the accumulation of inappropriately high levels of the CNS splice form in non-CNS tissues. The chromosomal Ddc transcript is similarly affected. These results show unexpected heterogeneity in the splicing of individual mRNAs as a response to heat shock and suggest that the Ddc CNS-specific splicing pathway is the default.

Alternative Splicing↗
PubMed1989

Molecular cloning and sequencing of a cDNA of rat dopa decarboxylase: partial amino acid homologies with other enzymes synthesizing catecholamines.

Dopa decarboxylase (DDC; aromatic-L-amino-acid decarboxylase; aromatic-L-amino-acid carboxylase, EC 4.1.1.28) was purified from rat liver and its partial sequence was determined. Synthetic oligonucleotides were used to construct and screen rat liver cDNA libraries, and three clones were isolated and sequenced. The 2 kilobases of DDC cDNA cloned consisted of a 5'-noncoding segment of 78 nucleotides, a coding region of 1440 nucleotides, and a 3'-noncoding region of 438 nucleotides. The encoded protein of 480 amino acid residues had a molecular weight of 54,000. A special feature of the primary structure of rat DDC was a repeating structure consisting of 29 amino acid residues. A sequence of 58 amino acid residues, including this repeating structure of rat DDC, was found to show homologies with those of rat tyrosine hydroxylase, human dopamine beta-hydroxylase, and bovine phenylethanolamine N-methyltransferase, other mammalian enzymes that synthesize catecholamines. These results indicate that catecholamine biosynthetic enzymes are structurally related and suggest that their homologous domains are important for catechol-protein interactions.

Amino Acid Sequence↗
PubMed1986

Differential responses of the dopa decarboxylase gene to 20-OH-ecdysone in Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) of Drosophila melanogaster responds to 20-OH-ecdysone in the mature larval epidermis and in imaginal discs (presumptive adult epidermis) in a tissue-specific manner. Exposure of the mature larval epidermis to 20-OH-ecdysone caused a rapid accumulation of DDC transcripts. In the absence of protein synthesis, transcript accumulation was substantially reduced suggesting an indirect hormonal effect on DDC transcription (and/or RNA turnover). By contrast, neither DDC activity induction nor transcript accumulation was detected in imaginal discs cultured in the continuous presence of the hormone. However, when discs were exposed to 20-OH-ecdysone and then cultured in its absence, DDC activity and DDC transcript levels started to increase 6 hr after hormone withdrawal. A Northern analysis failed to reveal any novel transcripts in discs making the utilization of an alternative promotor an unlikely explanation for the very different responses of the Ddc gene in the two epidermal tissues. The results demonstrate that the Ddc gene in the larval epidermis responds rapidly to an increase in hormone titer. In imaginal discs a fall in hormone titer is required before DDC transcripts accumulate.

Animals↗
PubMed1983

The cloned dopa decarboxylase gene is developmentally regulated when reintegrated into the Drosophila genome.

The Drosophila dopa decarboxylase gene, Ddc, functions normally when reintroduced into flies. DNA containing a cloned Ddc gene inserted into a P element transposon was injected into early embryos. Transformants were identified by suppression of the cuticular phenotype of a Ddc mutant allele. The reintegrated genes are expressed in the proper tissue and at the proper stages during development even though their positions within the genome are different from that of the wild-type Ddc gene. Absolute levels of DDC enzyme activity are within 35% of that found in wild-type Canton S flies, the source of the transforming DNA. The transformants' Ddc RNA is indistinguishable from that of wild type. One reintegrated Ddc gene, inserted on the X chromosome, is affected by the dosage compensation mechanism that leads to sex-specific differences in the expression of many X-chromosome genes.

Animals↗
PubMed1982

The prognostic and biological significance of cellular heterogeneity in medullary thyroid carcinoma: a study of calcitonin, L-dopa decarboxylase, and histaminase.

We evaluated the cellular distribution of calcitonin (CT), L-dopa decarboxylase (DDC), and histaminase [diamine oxidase (DAO)] in 33 patients with medullary thyroid carcinoma (MTC). CT immunostaining was uniform (greater than 90% of the cells) and intense (4+) in lesions from 7 patients with C-cell hyperplasia and 6 with microscopic MTC: conversely, patchy CT staining (less than 40% of the cells) and diminished intensity (1+) were found in metastases from 8 patients who died of virulent disease. In 17 patients who underwent thyroidectomy for macroscopic cervical MTC with no evidence distant metastases, CT staining was intense (3-4+) and homogeneous (greater than 90%) in 11 subjects who were well 0.5-16 yr postoperatively. Six patients with similar clinical presentations had heterogeneous staining for CT in primary tumor (less than 40% of the cells; 1-2+ intensity); 5 died of disseminated MTC 0.5-5 yr postoperatively (P less than 0.001). Biochemical studies of distant metastases revealed an inverse relationship between the distribution of CT and that of both relationship between the distribution of CT and that of both DDC (r = 0.71; P less than 0.01) and DAO (r = 0.81; P less than 0.001). We conclude that cellular heterogeneity in MTC tissue is associated with a distinct biochemical pattern, and its presence, whether in a primary or metastatic lesion, indicates a virulent neoplasm associated with a grave prognosis.

Adult↗
PubMed1970

Parkinson's disease: activity of L-dopa decarboxylase in discrete brain regions.

The activity of L-dopa decarboxylase was greatly reduced in the striatum, less so in the hypothalamus, and unchanged in the cortex of brains of patients with Parkinson's disease. However, it appears that even in the striatum enough activity remained to allow for the formation of dopamine from L-dopa in patients treated with large doses of L-dopa.

Basal Ganglia↗