Molecular characterization and expression analysis of dopa decarboxylase involved in the antibacterial innate immunity of the freshwater crayfish, Procambarus clarkii.

Dopa decarboxylase (DDC) is responsible for the synthesis of dopamine, which acts as an important modulator in the nervous systems of vertebrates and invertebrates. Recent studies have indicated that DDC also plays crucial roles in the insect innate immune system. However, the functions of DDC in immunomodulation in crustaceans have not been thoroughly elucidated to date. In this study, a new full-length cDNA of the DDC protein was identified from red swamp crayfish, Procambarus clarkii (named Pc-ddc). The ORF of Pc-ddc encoded 474 amino acids, which possessed a 377-amino-acid domain. Pc-ddc was expressed at a relatively high level in the hemocytes and gills of crayfish. This protein was expressed at a relatively low level in the hepatopancreas and intestine. The expression level of Pc-ddc was clearly upregulated in hemocytes, hepatopancreas, gills, and intestine tissues after challenge with S. aureus or E. ictaluri. The results of the enzyme catalysis assay showed that the enzyme catalysis activity of rPc-DDC was 35 ±â€¯2.8 ng h-1 mg-1 (n = 3). In addition, the results of the mimetic crayfish hemocytes encapsulation assay showed that the encapsulation rate of beads coated with rPc-DDC was clearly increased. The results of the bacterial binding assay showed that rPc-DDC strongly binds to S. aureus and E. ictaluri. Finally, when Pc-ddc was knocked down, the number of surviving crayfish clearly decreased after S. aureus or E. ictaluri was injected. All of these results indicate that Pc-DDC is an important immunomodulating enzyme in the neuroendocrine-immune (NEI) system of crayfish.

Histidine decarboxylase, DOPA decarboxylase, and vesicular monoamine transporter 2 expression in neuroendocrine tumors: immunohistochemical study and gene expression analysis.

Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.

Dopa decarboxylase: a model gene-enzyme system for studying development, behavior, and systematics.

Throughout its long evolutionary history, the Dopa decarboxylase gene (Ddc) has acquired a variety of functions in insects. The enzyme (DDC) catalyzes the production of the neural transmitters dopamine and serotonin. Not surprisingly, evidence of the enzyme's involvement in the behavior of insects is beginning to accumulate. In addition, DDC plays a role in wound healing, parasite defense, pigmentation, and cuticle hardening. A high degree of sequence conservation has allowed comparisons of the Ddc-coding regions from various insects, facilitating a number of recent studies on insect systematics. This review outlines the diverse functions of Ddc and illustrates how studies of this model system address many questions on insect neurobiology, developmental biology, and systematics.

Monoamine- and histamine-synthesizing enzymes and neurotransmitters within neurons of adult human cardiac ganglia.

BACKGROUND: Cardiac ganglia were originally thought to contain only cholinergic neurons relaying parasympathetic information from preganglionic brain stem neurons to the heart. Accumulating evidence, however, suggests that cardiac ganglia contain a heterogeneous population of neurons that synthesize or respond to several different neurotransmitters and neuropeptides. Reports regarding monoamine and histamine synthesis and neurotransmission within cardiac ganglia, however, present conflicting information or are limited in number. Furthermore, very few studies have examined the neurochemistry of adult human cardiac ganglia. The purpose of this study was, therefore, to determine whether monoamine- and histamine-synthesizing enzymes and neurotransmitters exist within neurons of adult human cardiac ganglia. METHODS AND RESULTS: Human heart tissue containing cardiac ganglia was obtained during autopsies of patients without cardiovascular pathology. Avidin-biotin complex immunohistochemistry was used to demonstrate tyrosine hydroxylase, L-dopa decarboxylase, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, tryptophan hydroxylase, and histidine decarboxylase immunoreactivity within neurons of cardiac ganglia. Dopamine, norepinephrine, serotonin, and histamine immunoreactivity was also found in ganglionic neurons. Omission or preadsorption of primary antibodies from the antisera and subsequent incubation with cardiac ganglia abolished specific staining in all cases examined. CONCLUSIONS: Our results suggest that neurons within cardiac ganglia contain enzymes involved in the synthesis of monoamines and histamine and that they contain dopamine, norepinephrine, serotonin, and histamine immunoreactivity. Our findings suggest a putative role for monoamine and histamine neurotransmission within adult human cardiac ganglia. Additional, functional evidence will be necessary to evaluate what the physiological role of monoamines and histamine may be in neural control of the adult human heart.

Distribution and origin of aminergic neurones in dog small intestine.

In dog ileum, axons containing immunoreactivity for tyrosine hydroxylase (TH) and for DOPA decarboxylase (DDC) invest the neurones of the enteric ganglia and are present around arterioles, in the deep plexus of the circular muscle and in the laminar propria of the mucosal villi. Immunoreactivity for serotonin (5-HT) was present in mucosal enterochromaffin cells and in varicose axons investing cells of the submucous plexus, but not in axons in myenteric plexus, circular muscle or mucosa. No enteric neuronal cell bodies were immunoreactive for DDC or for 5-HT. Two weeks after extrinsic denervation of the ileum, all TH staining was absent. By contrast, the pattern of DDC staining was unchanged, except around blood vessels. We conclude that motility of dog ileum is likely to be regulated by sympathetic noradrenergic inputs and by intrinsic serotonergic and 'amine-handling' neurones. Dopaminergic innervation of dog ileum appears to be restricted to a sparse vasomotor supply.

Sympathetic innervation of the liver in man and dog: an immunohistochemical study.

The sympathetic innervation of human and dog livers was examined by immunohistochemical localization of neuron-specific enolase to visualize the total complement of hepatic nerves and the localization of two enzymes involved in catecholamine synthesis, tyrosine hydroxylase and dihydroxyphenylalanine decarboxylase, to visualize sympathetic nerves. Similar results were obtained for both man and dog. About 60% of the non-myelinated axons supplying the hepatic parenchyma, and virtually all those supplying the vasculature, appeared to be sympathetic. The pattern of dihydroxyphenylalanine decarboxylase immunoreactivity was compatible with innervation of the intrahepatic hepatic arteries and portal veins by dopaminergic as well as by noradrenergic sympathetic nerves. By contrast, there was no evidence for a dopaminergic component in the parenchymal sympathetic innervation.

Neuronal metabolism and DOPA decarboxylase immunoreactivity in terminal noradrenergic sympathetic axons of rat.

This study was undertaken to determine whether immuno-histochemical staining for DOPA decarboxylase (DDC) is present in axons of rat noradrenergic sympathetic neurons. A sparse plexus of varicose axons exhibiting DDC-like immunoreactivity (DDC-IR) was associated with blood vessels and acini in the submandibular gland, but this was much less extensive than the population that exhibited tyrosine hydroxylase-like immunoreactivity (TH-IR). The varicose terminal TH-IR axons in atrium, spleen, and vas deferens were devoid of DDC-IR both in grown rats and during the post-natal period of axon growth, although weak DDC-IR was seen in large pre-terminal nerve bundles. Similar patterns of staining were seen with paraffin-embedded and with frozen, formaldehyde-fixed material. No enhancement of DDC-IR was seen in any tissue after chronic alteration of catecholamine turnover with reserpine or alpha-methyl-para-tyrosine, and the numbers of submandibular DDC-IR axons were not increased by disruption of axonal transport with colchicine or by decentralization of the superior cervical ganglion. We conclude that terminal noradrenergic axons contain insufficient DDC-IR for microscopic visualization, regardless of their metabolic state, reinforcing previous evidence that DDC-IR can be used as a histochemical marker for dopaminergic axons. By this criterion, the rat submandibular gland may receive a sparse dopaminergic innervation.

Immuno-cross-reactivity of histidine and dopa decarboxylases.

Immuno-cross-reactivity between histidine decarboxylase (HDC) and dopa decarboxylase (DDC) was investigated. By comparing the cDNA sequences of rat HDC with rat and guinea-pig DDCs, we found a region that may possibly be related to the cross-reactivity of anti-rat HDC antibody with guinea-pig DDC. The peptide encoded by this region was synthesized and anti-peptide antibody was prepared. We also purified HDC and DDC homogeniously from fetal rat liver and guinea-pig liver, respectively. On immunoblotting, anti-peptide antibody recognized both rat HDC and guinea-pig DDC. Anti-HDC polyclonal antibody which also recognizes both enzymes detected only rat HDC when it was absorbed by the peptide. This result indicates that this region is responsible for the immuno-cross-reactivity of anti-rat HDC antibody with guinea-pig DDC.

Molecular cloning of guinea-pig aromatic-L-amino acid decarboxylase cDNA.

Guinea-pig aromatic-L-amino acid decarboxylase (DOPA decarboxylase, DDC), but not rat DDC, reacts with the antibody against rat histidine decarboxylase (HDC). For determination of the molecular reaction for this cross-reactivity, a cDNA clone of guinea-pig DDC was isolated. Guinea-pig DDC consists of 480 amino acids and its molecular weight is 54,148. The sequence identify of guinea-pig DDC with rat DDC is 86%. Guinea-pig DDC has a region showing 100% sequence identity with rat HDC, but only 67% sequence identity with rat DDC, suggesting that this region is related with the cross-reactivity of guinea-pig DDC and anti-rat HDC antibody.

Purification and characterization of L-dopa decarboxylase from human kidney.

L-dopa decarboxylase has been purified to homogeneity from post mortem removed human kidneys. Homogeneity was examined by polyacrylamide gel electrophoresis (PAGE) analysis both in the presence and absence of SDS. The enzyme has a molecular weight of 100,000 daltons estimated by gel filtration and 50,000 daltons determined after SDS-PAGE. Human L-dopa decarboxylase therefore is a dimer. Polyclonal antibodies produced against human L-dopa decarboxylase react with the 50,000 daltons enzyme subunit after immuno-blotting and also precipitates enzyme activity. Activity against L-dopa is partially inhibited by 5-hydroxytryptophan (5-HTP). The effect of various cations on L-dopa decarboxylase activity has also been tested.

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.

The innervation of the renal cortex in the dog. An ultrastructural study.

Two cytochemical techniques were used at the ultrastructural level to study the distribution of specific axon types to different intrarenal structures in the dog. Using the chromaffin reaction to distinguish catecholaminergic fibres from other axon populations, it was found that the renal cortex of the dog is supplied only by catecholaminergic nerves. Immunostaining for tyrosine hydroxylase (TH) labelled all of the intracortical nerves, and 20% to 25% of these profiles also contained dopa decarboxylase (DDC)-immunoreactivity, indicating they were dopaminergic rather than noradrenergic. Both DDC-positive and DDC-negative axons were seen in close association (approximately 80 nm) with blood vessels and juxtaglomerular cells as well as tubular epithelial cells. The distribution of TH- and DDC-immunoreactive nerves in the renal cortex is compatible with existing functional evidence indicating that both dopaminergic and noradrenergic nerves are involved in the regulation of renal blood flow, tubular reabsorption and renin release.

Two classes of sympathetic nerves with different dopa decarboxylase immunoreactivities exist in dog vas deferens.

To determine whether dihydroxyphenylalanine (DOPA) decarboxylase (DDC) activity in the terminal regions of noradrenergic axons varies with axonal length, we compared the pattern of immunohistochemical staining for DDC in the 'short' terminal nerves of dog vas deferens with that in the 'long' nerves of spleen and atrium. The terminal nerves supplying the muscular coats of the vas deferens were, like those in spleen and heart, devoid of DDC immunoreactivity. The presence of this enzyme is therefore not characteristic of either short or long noradrenergic axons, in support of previous evidence that it is a specific marker for dopaminergic terminal nerves. Many axons supplying the mucosal epithelial cells in the vas deferens were DDC-positive, suggesting the existence of a dopaminergic innervation.

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.

Demonstration of immunohistochemical and immunochemical cross-reactivity of L-histidine and L-dopa decarboxylases using antibodies against the two enzymes.

Both rat L-histidine decarboxylase (HDC) and guinea-pig L-DOPA decarboxylase (DDC) were shown immunohistochemically and immunochemically to react with anti-rat HDC antibody. No cross-reaction was observed in immunoprecipitation experiments, but both anti-rat HDC antibody and anti-rat DDC antibody immunostained neurons in the substantia nigra, raphe nucleus and locus coeruleus of guinea-pig brain. Moreover, on immunoblotting, anti-rat HDC antibody recognized not only rat HDC but also guinea-pig DDC, but not rat DDC. However, anti-rat DDC antibody showed no immunohistochemical or immunochemical cross-reactivity with rat HDC.

Immunohistochemical analysis of the cross-reaction of anti-rat histidine decarboxylase antibody with guinea-pig DOPA decarboxylase.

L-Histidine decarboxylase [L-histidine carboxylyase, HDC, EC 4.1.1.22] is an enzyme distinct from L-DOPA decarboxylase [L-aromatic amino acid carboxylyase, DDC, EC 4.1.1.28]: the two decarboxylases from fetal rat liver were completely separated from each other by DEAE-cellulose column chromatography and by affinity chromatography with L-carnosine as a ligand. The antibody raised against this HDC inhibited the HDC's from rat and guinea-pig brains very strongly, but their DDCs very weakly. However, in immunofluorescent histochemical studies, the antibody cross-reacted with DDC-like immunoreactive structures, such as chromaffin cells of the adrenal medulla, the raphe nucleus, the substantia nigra, and the locus coeruleus of the brain of guinea-pigs, but not of rats, suggesting that these two decarboxylases share some antigenic structures.

Some immunochemical properties of pig kidney DOPA decarboxylase.

Antibodies raised in rabbits against pig kidney DOPA decarboxylase show immunological cross-reactivity towards extracts from monkey, beef, rat and rabbit kidney. The influence of the immuno-reaction on the enzymatic activity has been investigated.

A novel form of DOPA decarboxylase produced in drosophila cells in response to 20-hydroxyecdysone.

Two cloned derivatives of the Kc cell line of Drosophila were shown to produce DOPA decarboxylase following administration of the steroid moulting hormone 20-hydroxyecdysone. In the continuous presence of the hormone at a concentration of 2 X 10(-7) M, DOPA decarboxylase activity first appeared between 48 and 72 h. Because of this lag, the tissue culture system promises to serve as a useful model for those in vivo situations where increases in the hormone titre precede increases in DOPA decarboxylase activity. In clone 7C4, after maximal enzyme activity was achieved at 144 h, the enzyme activity per cell decreased as the cells resumed division following the hormone-induced division arrest. In clone 7E10, cell division never resumed in the presence of 20-hydroxyecdysone and DOPA decarboxylase activity per cell increased continuously from the time it first appeared. When line 7E10 was exposed to a 6-h pulse of the steroid, enzyme activity appeared about 18 h earlier than in the presence of continuous hormone and, further, the cells were released from division arrest. Enzyme activity per cell then declined from an early 96-h maximum. The enzyme produced by the cell lines was immunologically distinct from the enzyme produced in vivo and ion-exchange column chromatography resolved the enzyme from cells and intact organisms into two species.(ABSTRACT TRUNCATED AT 250 WORDS)