Healing of Ocular Herpetic Disease Following Treatment With an Engineered FGF-1 Is Associated With Increased Corneal Anti-Inflammatory M2 Macrophages.

Herpes simplex virus 1 (HSV-1) infects the cornea and caused blinding ocular disease. In the present study, we evaluated whether and how a novel engineered version of fibroblast growth factor-1 (FGF-1), designated as TTHX1114, would reduce the severity of HSV-1-induced and recurrent ocular herpes in the mouse model. The efficacy of TTHX1114 against corneal keratopathy was assessed in B6 mice following corneal infection with HSV-1, strain McKrae. Starting day one post infection (PI), mice received TTHX1114 for 14 days. The severity of primary stromal keratitis and blepharitis were monitored up to 28 days PI. Inflammatory cell infiltrating infected corneas were characterized up to day 21 PI. The severity of recurrent herpetic disease was quantified in latently infected B6 mice up to 30 days post-UVB corneal exposure. The effect of TTHX1114 on M1 and M2 macrophage polarization was determined in vivo in mice and in vitro on primary human monocytes-derived macrophages. Compared to HSV-1 infected non-treated mice, the infected and TTHX1114 treated mice exhibited significant reduction of primary and recurrent stromal keratitis and blepharitis, without affecting virus corneal replication. The therapeutic effect of TTHX1114 was associated with a significant decrease in the frequency of M1 macrophages infiltrating the cornea, which expressed significantly lower levels of pro-inflammatory cytokines and chemokines. This polarization toward M2 phenotype was confirmed in vitro on human primary macrophages. This pre-clinical finding suggests use of this engineered FGF-1 as a novel immunotherapeutic regimen to reduce primary and recurrent HSV-1-induced corneal disease in the clinic.

Nerve growth factor nonresponsive pheochromocytoma cells: altered internalization results in signaling dysfunction.

Variant rat pheochromocytoma (PC12) cells which fail to respond to nerve growth factor (NGF) (PC12nnr5) (Green, S. H., R. E. Rydel, J. L. Connoly, and L. A. Greene. 1986. J. Cell Biol. 102:830-843) bind NGF at both high and low affinity sites. Although still undefined at the molecular level, these have been referred to as type I (high) and type II (low) receptors. They are apparently composed of two membrane-bound proteins, p75 and the protooncogene trk, both of which bind NGF, and apparently contribute singularly or in concert to the two observed affinities, and to the promotion of the NGF effects. In native PC12 cells, only the high affinity receptors are apparently capable of mediating internalization and degradation. PC12nnr5 cells also display type I binding, but the subsequent internalization is not the same fashion as in the parental cell line, nor is it subjected to lysosomal degradation. Rather it is initially sequestered during the first 15 min, and is eventually released intact into the medium. In contrast, EGF is bound, internalized, and degraded by PC12nnr5 cells, albeit less efficiently than in the parent cells. These observations argue that the defect(s) preventing the PC12nnr5 variants from responding to NGF prevents competent internalization, which in the case of NGF, may be required for the full expression of activity. The absence of trk, as one alteration in PC12nnr5 cells (Loeb, D. M., J. Maragos, D. Martin-Zanca, M. V. Chao, L. F. Parada, and L. A. Greene. 1991. Cell. 66:961-966), is consistent with this conclusion.

Internalization and cycling of nerve growth factor in PC12 cells: interconversion of type II (fast) and type I (slow) nerve growth factor receptors.

The effects of agents that inhibit receptor-mediated endocytosis on type I (slow or high-affinity) and type II (fast or low-affinity) NGF binding have been examined in rat PC12 cells. Compounds interfering with endocytosis eliminate type I NGF binding; those interfering with acidification of endosomal vesicles cause increased type I binding at the expense of type II binding. Measurement of NGF binding during and after treatment with inhibitors indicates that NGF receptors rapidly cycle from the cell surface into an undefined endocytotic compartment and back to the surface with little degradation of receptor or NGF, consistent with a model in which NGF receptors are rapidly and reversibly endocytosed or sequestered; those receptors free on the surface represent type II NGF receptors, while those in the process of endocytosis represent type I NGF receptors. The type I and type II NGF receptor species can be interconverted by agents that can manipulate the position of the receptor in the internalization cycle.

Evidence for evolutionary duplication of genes in the dopa decarboxylase region of Drosophila.

The region surrounding the dopa decarboxylase gene (Ddc) of Drosophila contains a cluster of genes, many of which appear to be functionally related by virtue of their effects on cuticle development and/or catecholamine metabolism. In this report we describe evidence that the Ddc gene and the closely linked alpha-methyldopa hypersensitive (amd) gene share extensive sequence homology and are the products of a gene duplication event. The two genes are transcribed convergently and are separated by 2.4 kb. A gene located between Ddc and amd expresses a 2.0-kb mRNA and appears to partially overlap the Ddc gene. The organization of these transcripts implies a complex series of events giving rise to the present pattern. The patterns of expression of these genes do not support a model of coordinate regulation, but are more consistent with a pattern of duplication and divergence to various related metabolic subspecialties. These data provide the first evidence for structural relationships among genes in the 37C cluster.

Time-related neuronal changes following middle cerebral artery occlusion: implications for therapeutic intervention and the role of calpain.

Changes in neocortex and striatum were characterized over time following focal ischemia to the brain. Rats were subjected to permanent middle cerebral artery occlusion (MCA-O) and sacrificed 1, 3, 6, 12, or 24 h later. The affected tissue was processed for tetrazolium chloride (TTC) and cresyl violet staining, as well as for Western blots to detect calpain-induced spectrin proteolysis. Significant changes in cell size and spectrin breakdown occurred within the first hour of occlusion, with further, dramatic changes in these two early markers continuing over time. Initial evidence of cell loss was noted at 1 h postocclusion in the striatum and at 3 h in the neocortex. However, even in the center of the most affected portion of the neocortex, the majority of cells appeared to be intact through 6 h. By this time, a significant TTC-defined infarct also emerged. These quantitative data indicate that calpain-induced proteolysis occurs very soon after the ischemic insult, is correlated with earliest changes in cell hypotrophy, and precedes or occurs in tandem with evidence of significant cell loss. They also demonstrate that, while some cell loss occurs earlier than previously believed, the majority of cells remains morphologically intact well beyond what is typically thought to be the window of opportunity for intervention. The results thus raise the question of how long after the ischemic event pharmaceutic intervention might be employed to salvage substantial numbers of neurons.

Local cerebral glucose utilization and cytoskeletal proteolysis as indices of evolving focal ischemic injury in core and penumbra.

To ascertain the tempo of progression to irreversible injury in focal ischemia, we subjected halothane-anesthetized Sprague-Dawley rats to photochemically induced distal middle cerebral artery occlusion (dMCAO) combined with permanent ipsilateral and 1 h contralateral common carotid artery occlusions. Head temperature was maintained at 36 degrees C. At times centered at either 1.5 or 3 h post-dMCAO, the rate of local glucose metabolism (lCMRgl) was measured by 2-deoxyglucose autoradiography, and cytoskeletal proteolysis was assessed regionally by an immunoblotting procedure to detect spectrin breakdown products. At 1.5 h (n = 5), the cortical ischemic core was already severely hypometabolic (lCMRgl 15.5 +/- 10.8 mumol 100 g-1 min-1, mean +/- SD), whereas the cortical penumbral zone was hypermetabolic (69.0 +/- 9.7). (The lumped constant was verified to be unchanged by methylglucose studies). Neutral red pH studies at this time point showed that both the core and penumbral zones were equally acidotic. By 3 h post-dMCAO (n = 6), lCMRgl in the penumbral zone had fallen to low levels (15.4 +/- 2.2 mumol 100 g-1 min-1) equal to those of the ischemic core (16.7 +/- 4.5). Correspondingly, spectrin breakdown in the ischemic core was advanced at both 2 and 3.5 h post-dMCAO (36 +/- 18% and 33 +/- 18% of total spectrin, respectively), whereas in the penumbral zone spectrin breakdown was less extensive and more highly variable at both times (22 +/- 23% and 29 +/- 16%). We conclude that irreversible deterioration of the ischemic core, as evidenced by the onset of local cytoskeletal proteolysis, begins within 2 h of middle cerebral artery occlusion. In the ischemic penumbra, the transition from glucose hyper- to hypometabolism occurs by 3.5 h and is associated with a milder and more variable degree of spectrin breakdown.

Peptide alpha-keto ester, alpha-keto amide, and alpha-keto acid inhibitors of calpains and other cysteine proteases.

A series of dipeptidyl and tripeptidyl alpha-keto esters, alpha-keto amides, and alpha-keto acids having leucine in the P2 position were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, cathepsin B, and papain. In general, peptidyl alpha-keto acids were more inhibitory toward calpain I and II than alpha-keto amides, which in turn were more effective than alpha-keto esters. In the series Z-Leu-AA-COOEt, the inhibitory potency decreased in the order: Met (lowest KI) > Nva > Phe > 4-Cl-Phe > Abu > Nle (highest KI) with calpain I, while almost the reverse order was observed for calpain II. Extending the dipeptide alpha-keto ester to a tripeptide alpha-keto ester yielded significant enhancement in the inhibitory potency toward cathepsin B, but smaller changes toward the calpains. Changing the ester group in the alpha-keto esters did not substantially decrease KI values for calpain I and calpain II. N-Monosubstituted alpha-keto amides were better inhibitors than the corresponding alpha-keto esters. alpha-Keto amides with hydrophobic alkyl groups or alkyl groups with an attached phenyl group had the lower KI values. N,N-Disubstituted alpha-keto amides were much less potent inhibitors than the corresponding N-monosubstituted peptide alpha-keto amides. The peptide alpha-keto acid Z-Leu-Phe-COOH was the best inhibitor for calpain I (KI = 0.0085 microM) and calpain II (KI = 0.0057 microM) discovered in this study. It is likely that the inhibitors are transition-state analogs and form tetrahedral adducts with the active site cysteine of cysteine proteases and form hydrogen bonds with the active site histidine and possibly another hydrogen bond donor in the case of monosubstituted amides. Several inhibitors prevented spectrin degradation in a platelet membrane permeability assay and may be useful for the treatment of diseases which involve neurodegeneration.

Novel peptidyl alpha-keto amide inhibitors of calpains and other cysteine proteases.

A series of new dipeptidyl alpha-keto amides of the general structure R1-L-Leu-D,L-AA-CONH-R2 were synthesized and evaluated as inhibitors for the cysteine proteases calpain I, calpain II, and cathepsin B. They combine 10 different N-protecting groups (R1), 3 amino acids residues in P1 (AA), and 44 distinct substituents on the alpha-keto amide nitrogen (R2). In general, calpain II was more sensitive to these inhibitors than calpain I, with a large number of inhibitors displaying dissociation constants (Ki) in the 10-100 nM range. Calpain I was also effectively inhibited, but very low Ki values were observed with a smaller number of inhibitors than with calpain II. Cathepsin B was weakly inhibited by most compounds in this study. The best inhibitors for calpain II were Z-Leu-Abu-CONH-CH2-CHOH-C6H5 (Ki = 15 nM), Z-Leu-Abu-CONH-CH2-2-pyridyl (Ki = 17 nM), and Z-Leu-Abu-CONH-CH2-C6H3(3,5(OMe)2) (Ki = 22 nM). The best calpain I inhibitor in this study was Z-Leu-Nva-CONH-CH2-2-pyridyl (Ki = 19 nM). The peptide alpha-keto amide Z-Leu-Abu-CONH-(CH2)2-3-indolyl was the best inhibitor for cathepsin B (Ki = 31 nM). Some compounds acted as specific calpain inhibitors, with comparable activity on both calpains I and II and a lack of activity on cathepsin B (e.g., 40, 42, 48, 70). Others were specific inhibitors for calpain I (e.g., 73) or calpain II (e.g., 18, 19, 33, 35, 56). Such inhibitors may be useful in elucidating the physiological and pathological events involving these proteases and may become possible therapeutic agents.

Synthesis and biological activity of N6-(p-sulfophenyl)alkyl and N6-sulfoalkyl derivatives of adenosine: water-soluble and peripherally selective adenosine agonists.

A series of N6-(p-sulfophenyl)alkyl and N6-sulfoalkyl derivatives of adenosine was synthesized, revealing that N6-(p-sulfophenyl)adenosine (10b) is a moderately potent (Ki vs [3H]PIA in rat cortical membranes was 74nM) and A1-selective (120-fold) adenosine agonist, of exceptional aqueous solubility of > 1.5 g/mL (approximately 3 M). Compound 10b was very potent in inhibiting synaptic potentials in gerbil hippocampal slices with an IC50 of 63 nM. At a dose of 0.1 mg/kg ip in rats, 10b inhibited lipolysis (a peripheral A1 effect) by 85% after 1 h. This in vivo effect was reversed using the peripherally selective A1-antagonist 1,3-dipropyl-8-[p-(carboxyethynyl)phenyl]xanthine (BW1433). The same dose of 10b in NIH Swiss mice (ip) was nearly inactive in locomotor depression, an effect that has been shown to be centrally mediated when elicited by lower doses of other potent adenosine agonists, such as N6-cyclohexyladenosine (CHA) (Nikodijevic et al. FEBS Lett. 1990, 261, 67). HPLC studies of biodistribution of a closely related and less potent homologue, N6-[4-(p-sulfophenyl)butyl]adenosine indicated that a 25 mg/kg ip dose in mice resulted in a plasma concentration after 30 min of 0.46 micrograms/mL and no detectable drug in the brain (detection limit < 0.1% of plasma level). Although 10b at doses > 0.1 mg/kg in mice depressed locomotor activity, this depression was unlike the effects of CHA and was reversible by BW1433. These data suggest that 10b is a potent adenosine agonist in vivo and shows poor CNS penetration.

Temporal ordering of pathogenic events following transient global ischemia.

Rats were subjected to transient global ischemia (four vessel occlusion) and time-related changes in the selectively vulnerable hippocampal field CA1 were characterized. The assessment included ex vivo field responses to afferent stimulation, silver staining, calpain-induced spectrin breakdown, chromatolysis, and cell death, beginning at 6 h post-ischemia and continuing until total disintegration of the pyramidal cells occurred several days later. The earliest change observed was a modest increase in the slope and amplitude of field CA1 potentials (at 6 h). The hyperresponsiveness was most apparent at higher stimulation currents and persisted unchanged at 16 h post-ischemia. Three effects became detectable within 24 h, post-ischemia: (a) an increase in concentrations of calpain-mediated, spectrin breakdown products; (b) enhanced silver staining in the deep pyramidal neurons of the field CA1 with lesser, though still apparent, staining of stratum radiatum, and (c) a decrease in amplitude and slope of field CA1 responses to afferent stimulation. Both the concentration of spectrin breakdown products and the intensity of silver staining progressively increased to a maximum at four days post ischemia, while the amplitude and slope of the field responses dropped to a very low level between 24 and 48 h. Disturbances of Nissl staining were finally evident at 48 h, with nearly complete disappearance of staining at five days post-ischemia. This study provides the first demonstration of a close and early temporal relationship between calpain proteolysis, subcellular damage to the pyramidal cells and their loss of function following global ischemia, prior to their eventual death.

I.c.v. infusions of ACPD(1S,3R) attenuate learning in a Morris water maze paradigm.

The functional role of metabotropic glutamate receptors is beginning to be understood. Cognitive effects of metabotropic receptor activation were examined in the present study in a Morris water maze following intraventricular (i.c.v.) infusions of ACPD(1S,3R). I.c.v. infusions of ACPD substantially increased the amount of time animals took to find a hidden platform in a water maze. Current results demonstrate that subtoxic doses of ACPD can produce in vivo effects with functional significance. Spatial learning abilities are impaired following activation of metabotropic receptors with ACPD(1S,3R). Negative implications are provided for the use of cognitive enhancing compounds that stimulate metabotropic glutamate receptors.

Attenuation of vasospasm and hemoglobin-induced constriction in the rabbit basilar artery by a novel protease inhibitor.

Calcium-activated proteolysis mediated by the protease inhibitor, calpain, has recently been implicated in the pathogenesis of cerebral vasospasm. The effect of one inhibitor of calcium-activated proteolysis, z-Leu-Phe-CONH-morpholene (zLF), on cerebrovascular constriction was examined in two experimental paradigms. In the first paradigm, the rabbit basilar artery (BA) was visualized via a transclival exposure, and its diameter was monitored using videomicroscopy. In the second experimental paradigm two intracisternal injections of autologous blood were administered to mimic a subarachnoid hemorrhage (SAH). The BA was visualized via the transclival exposure, and its luminal diameter was measured. Topical application of oxyhemoglobin (OxyHb), a known pathogenic agent in cerebral vasospasm, elicited vasoconstriction in normal animals, reducing arterial diameter to approximately 75% of resting levels. Pretreatment with zLF (100, 200, or 300 microM) attenuated vasoconstriction induced by OxyHb. In an experimental model of SAH, the diameter of the BA was reduced after the first injection of blood to approximately 67% of normal resting levels when measured 3 to 4 days later. This vasospastic response was reversed significantly by topical application of zLF (100 microM); vascular diameter was increased to approximately 84% of normal resting levels. These findings demonstrate that both acute OxyHb-induced constriction and blood-induced vasospasm are sensitive to an inhibitor of the proteolytic enzyme, calpain. Together, these observations indicate an important role for calcium-activated proteolysis in the development and maintenance of vasospasm after SAH. In addition, it may be inferred from the data that inhibitors of calcium-activated proteolysis may be useful therapeutic agents for treating this form of cerebrovascular disease.

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.

Overlapping transcription units in Drosophila: sequence and structure of the Cs gene.

The Cs gene lies between the functionally and evolutionarily related dopa decarboxylase (Ddc) and l(2)amd loci of Drosophila. The Cs and Ddc genes overlap at their 3' ends, implying that the transcription termination signals of these genes are polar, since each gene's primary transcript contains the complement of the other gene's transcription termination signals. The mature transcripts of the Cs and Ddc genes are complementary for a short distance and the primary transcripts may be complementary over thousands of base pairs. Despite intensive mutagenesis in this region, no mutations affecting the Cs transcript have been recovered although over 90 alleles of the two flanking genes (Ddc and l(2)amd) have been identified. Unlike the flanking Ddc and l(2)amd genes, the structure of the Cs gene and the temporal and tissue specificity of Cs expression are inconsistent with any structural or functional relatedness to the Ddc gene family. The internal structure of the Cs transcript is unlike that of most protein coding genes; it contains several open reading frames which are not situated favorably for efficient translation of the Cs message. This unusual internal structure may be the basis of the observed mutational silence of the Cs locus.

Modulation of growth factor induced fiber outgrowth in rat pheochromocytoma (PC12) cells by a fibronectin receptor antibody.

Rat pheochromocytoma PC12 cells respond to the binding of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) by extending neurites in a manner resembling sympathetic neurons. This response requires cell attachment to an appropriate substratum (Fujii et al., J. Neurosci., 2:1157, 1982); attachment factors which function in this capacity include the adhesive proteins fibronectin and laminin. Incubating PC12 cells with a polyclonal antiserum directed against a putative 140-kDa fibroblast cell surface fibronectin receptor (anti-gp140) perturbed spreading but not attachment of the cells to fibronectin and laminin substrates. However, in the presence of anti-gp 140 or its Fab fragments, NGF-stimulated neurite outgrowth was dramatically reduced. The antibody also caused a retraction of previously extended neurites. SDS-PAGE analysis of immunoprecipitates of PC12 cells surface labeled with 125I identified a prominent 120-140-kDa band, suggesting that the site of anti-gp140 action in PC12 cells is also through a fibronectin receptor.

v-src genes stimulate neurite outgrowth in pheochromocytoma (PC12) variants unresponsive to neurotrophic factors.

The NGF-nonresponsive rat pheochromocytoma PC12 variant nnr5, isolated by Green et al. (J Cell Biol 102:830-843, 1986), responds poorly or not at all to fibroblast growth factor. Transformation of PC-12nnr5 cells with v-src-expressing retroviruses results in vigorous neurite outgrowth, similar to that seen in the parent cell line. Thus though the PC12nnr5 cell line has a greatly impaired ability to respond to neurotrophic factors it still may extend neurites. This data is consistent with a model in which PC12nnr5 cells are unable to propagate intracellular second messengers, and this defect may be related to the expression of c-src gene products.

Differential induction of primary-response (TIS) genes in PC12 pheochromocytoma cells and the unresponsive variant PC12nnr5.

As a measure of the transmembrane signals that they transduce, two neurotrophic agents, nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), and the muscarinic agonist carbachol were compared for their ability to induce TIS (tetradecanoyl phorbol acetate-inducible sequences) transcripts, representing a family of immediate early response genes, in the rat pheochromocytoma cell line PC12 and the morphologically unresponsive variant PC12nnr5. Three genes, TIS1 (also designated NGFIB), TIS8 (also designated NGFIA), and TIS21, induced in these cells by NGF (Kujubu, D.A., Lim, R.W., Varnum, B.C., and Herschman, H.R. (1987) Oncogene 1, 257-262, 1987), are also induced by bFGF and carbachol. In native PC12 cells the level of expression of TIS8 and TIS21 is similar for all three stimuli, as well as for tetradecanoyl phorbol acetate (TPA). In contrast, the induction of TIS1 by NGF and TPA is slight and is only just detectable after stimulation by bFGF, but is strong for carbachol. Thus, although all of these agents can stimulate protein kinase (PK-C), at least one TIS gene can apparently be differentially regulated by these ligands, suggesting that alternative signaling pathways must also exist. In keeping with this view, bFGF, and to a lesser degree NGF, can elicit a TIS gene response in PC12 cells in which PK-C has been down-regulated with TPA. The response to carbachol (and TPA) is effectively blocked under these conditions. Since both NGF and bFGF stimulate neurite outgrowth in such cells, PK-C is apparently not essential, i.e. does not represent the sole mechanism, for signal transduction leading to modulation of gene expression for these factors. Consistent with this model, putative protein kinase inhibitors, K252a and sphingosine, did not inhibit the TIS gene responses to bFGF. However, these agents also failed to block TIS gene responses to carbachol and TPA indicating that they were ineffective as PK-C inhibitors under these conditions. The NGF-induced response was, however, blocked by K252a indicating a unique step in the mechanism of this factor not shared by the other ligands. Sphingosine did not block TIS induction with NGF. The mutant cell line PC12 nnr5 does not respond morphologically to either NGF or bFGF. However, TIS gene responses to bFGF are unaffected, whereas those to NGF are completely abolished. The response to TPA is altered quantitatively but not qualitatively; the induction by carbachol is largely eliminated, apparently as a result of a 90% reduction in muscarinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Bioassay detection of mouse nerve growth factor (mNGF) in the brain of adult mice.

Two pools of seven brains each from adult Swiss-Webster mice were homogenized, and supernatants were collected for bioassay. PC-12 cells were placed in a bioassay plate at time zero, at a concentration of 10(4) cells per well, and primed for 48 hours in a medium containing 50 ng/ml of mNGF. The PC-12 cell bioassay for neurite outgrowth was conducted after primed cells were exposed to an NGF-free medium for 24 hours. Suitable controls for serum toxicity and cell viability were established. The sensitivity of the bioassay approximates 100 pg NGF/ml. The results showed 80-100% neurite outgrowth in wells exposed to brain pool supernatant (BPS) alone, and control level outgrowth (3-8%) in wells containing BPS and specific anti-beta-NGF antibody. Therefore, the brains of Swiss-Webster adult mice contain an NGF-like substance which promotes neurite outgrowth in PC-12 cells. The substance probably is NGF itself, since the effect is blocked by specific NGF antiserum.

Sequence and structure of the dopa decarboxylase gene of Drosophila: evidence for novel RNA splicing variants.

In Drosophila, dopa decarboxylase (DDC) serves a dual role in neurotransmitter production and sclerotization of the cuticle. The Ddc gene is under complex hormonal and tissue-specific control and several sizes of Ddc RNA are observed at embryonic hatching, pupariation and adult eclosion. We present here the complete nucleotide sequence of the Drosophila dopa decarboxylase gene and the partial sequence of two corresponding Ddc cDNAs. The sequence allows us to account for the detailed structure of four of the five major Ddc RNA species observed. The cDNA sequence reveals the existence of previously undetected splicing events and provides evidence for two RNA splicing alternatives which appear to encode two protein isoforms. The structure, processing and developmental regulation of the Ddc transcripts and putative protein isoforms are discussed. Interestingly, the pyridoxal-binding peptide of porcine DDC matches the Drosophila sequence perfectly suggesting considerable selective pressure on at least portions of the sequence. This is the first available Ddc gene sequence from any organism and should serve as a basis of comparison for the related proteins of other species.