Chromatographic characterisation of 11 phytocannabinoids: Quantitative and fit-to-purpose performance as a function of extra-column variance.

INTRODUCTION: Cannabis sativa L. (cannabis) is utilised as a therapeutic and recreational drug. With the legalisation of cannabis in many countries and the anticipated regulation of potency that will accompany legalisation, analytical testing facilities will require a broadly applicable, quantitative, high throughput method to meet increased demand. Current analytical methods for the biologically active components of cannabis (phytocannabinoids) suffer from low throughput and/or an incomplete complement of relevant phytocannabinoids. OBJECTIVE: To develop a rapid, quantitative and broadly applicable liquid chromatography-tandem mass spectrometry analytical method for 11 phytocannabinoids in cannabis with acidic and neutral character. METHODOLOGY: Bulk diffusion coefficients were calculated using the Taylor-Aris open tubular method, with four reference compounds used to validate the experimental set-up. Three columns were quantitatively evaluated using van Deemter plots and fit-to-purpose performance metrics. Low (1.2 µL2 ) and standard (3.6 µL2 ) extra-column variance ultra-high pressure liquid chromatography (UPLC) configurations were contrasted. Method performance was demonstrated with methanolic cannabis flower extracts. RESULTS: Bulk diffusion coefficients and van Deemter plots for 11 phytocannabinoids are reported. The developed chromatographic method includes the challenging Δ8 /Δ9 -tetrahydrocannabinol isobars and, at 6.5 min, is faster than existing methods targeting similar panels of biologically active phytocannabinoids. CONCLUSIONS: The bulk diffusion coefficients and van Deemter curves informed the development of a rapid quantitative method and will facilitate potential expansion to include additional compounds, including synthetic cannabinoids. The developed method can be implemented with low or standard extra-column variance UPLC configurations.

Tapeworm larvae in Artemia franciscana (Crustacea: Anostraca) in the Godolphin lakes of Dubai (United Arab Emirates) throughout an annual cycle.

A total of 1840 brine shrimps (Artemia franciscana) were examined for cestode larvae at monthly intervals between November 2015 and June 2016. Of these, 663 (36.03%) specimens were infected with cysticercoids of seven cestode species in numbers between one and sixteen. During the first four months of examination, the percentage of infected shrimps was low but rose significantly with increasing temperatures in March, reaching maximum values in May. Flamingolepis liguloides and Flamingolepis flamingo showed the highest prevalence overall, at 25.3 and 10.7%, respectively. The intensity of infection was 1-10 and 1-4 cysticercoids, respectively. Eurycestus avoceti, Wardium stellorae, Gynandrotaenia stammeri, Anomotaenia tringae and Confluaria podicipina occurred at lower prevalence of 4.5, 3.2, 1.7, 0.3 and 0.05%, respectively. Up to four species were detected in one host.

Decreased cerebral spinal fluid neurotransmitter levels in Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive, multiple congenital anomaly syndrome with cognitive impairment and a distinct behavioral phenotype that includes autistic features. SLOS is caused by a defect in 3ß-hydroxysterol Δ(7)-reductase which leads to decreased cholesterol levels and elevated cholesterol precursors, specifically 7- and 8-dehydrocholesterol. However, the pathological processes contributing to the neurological abnormalities in SLOS have not been defined. In view of prior data suggesting defects in SLOS in vesicular release and given the association of altered serotonin metabolism with autism, we were interested in measuring neurotransmitter metabolite levels in SLOS to assess their potential to be used as biomarkers in therapeutic trials. We measured cerebral spinal fluid levels of serotonin and dopamine metabolites, 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) respectively, in 21 SLOS subjects. Results were correlated with the SLOS anatomical severity score, Aberrant Behavior Checklist scores and concurrent sterol biochemistry. Cerebral spinal fluid (CSF) levels of both 5HIAA and HVA were significantly reduced in SLOS subjects. In individual patients, the levels of both 5HIAA and HVA were reduced to a similar degree. CSF neurotransmitter metabolite levels did not correlate with either CSF sterols or behavioral measures. This is the first study demonstrating decreased levels of CSF neurotransmitter metabolites in SLOS. We propose that decreased levels of neurotransmitters in SLOS are caused by a sterol-related defect in synaptic vesicle formation and that CSF 5HIAA and HVA will be useful biomarkers in development of future therapeutic trials.

Annually recurring parthenogenesis in a zebra shark Stegostoma fasciatum.

A zebra shark, Stegostoma fasciatum, held in captivity at the Burj Al Arab aquarium, produced embryos and pups in the absence of a male. A total of 15 pups were produced from eggs laid within the aquarium over a period of four consecutive years commencing 2007. Parthenogenesis was confirmed through DNA analysis for three pups sampled during the first two consecutive egg cycles and is presumed to be the method of reproduction responsible thereafter.

Ctf19p: A novel kinetochore protein in Saccharomyces cerevisiae and a potential link between the kinetochore and mitotic spindle.

A genetic synthetic dosage lethality (SDL) screen using CTF13 encoding a known kinetochore protein as the overexpressed reference gene identified two chromosome transmission fidelity (ctf) mutants, YCTF58 and YCTF26. These mutant strains carry independent alleles of a novel gene, which we have designated CTF19. In light of its potential role in kinetochore function, we have cloned and characterized the CTF19 gene in detail. CTF19 encodes a nonessential 369-amino acid protein. ctf19 mutant strains display a severe chromosome missegregation phenotype, are hypersensitive to benomyl, and accumulate at G2/M in cycling cells. CTF19 genetically interacts with kinetochore structural mutants and mitotic checkpoint mutants. In addition, ctf19 mutants show a defect in the ability of centromeres on minichromosomes to bind microtubules in an in vitro assay. In vivo cross-linking and chromatin immunoprecipitation demonstrates that Ctf19p specifically interacts with CEN DNA. Furthermore, Ctf19-HAp localizes to the nuclear face of the spindle pole body and genetically interacts with a spindle-associated protein. We propose that Ctf19p is part of a macromolecular kinetochore complex, which may function as a link between the kinetochore and the mitotic spindle.

Tetrahydrobiopterin deficiency in human rabies.

Rabies is a fatal viral encephalitis characterized by a clinically acute and progressive course. With rare exceptions, there is a discrepancy between clinical outcome and frank histological alterations in rabies. Investigators have postulated that rabies virus may modify neurotransmission through occupancy of cellular receptors or alteration of ion channels. We took advantage of these observations to improvise a successful therapy for rabies. The Milwaukee protocol ( www.mcw.edu/rabies ) was further modified to treat two German patients. We measured pterins and monoamine neurotransmitter metabolites in the CSF of patients with rabies by HPLC with electrochemical or fluorescent detection. We report loss of tetrahydrobiopterin (BH(4)) and associated pathological decrease of dopaminergic and serotoninergic neurotransmission in three successive patients with rabies. CSF levels of BH(4) and neurotransmitter metabolites increased in two patients who were supplemented. Our findings support the long-standing speculation of modified neurotransmission in the pathogenesis of rabies, but by another mechanism. Brain turnover of dopamine and serotonin is reduced following rabies-acquired BH(4) deficiency. Neuronal nitric oxide synthase is BH(4)-dependent and may also be involved, possibly causing cerebrovascular insufficiency in one patient. This work must be carefully replicated in animal models and future patients. We are cautiously optimistic at the prospect of readily available, metabolically specific, enteral therapy for rabies.

Exploiting the complete yeast genome sequence.

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.

Generalised cranial artery spasm in human rabies.

In 2004, a teenager survived bat-associated rabies through the Milwaukee protocol (MP). This survivor and another patient with dog-associated rabies were found to have developed deficiencies of tetrahydrobiopterin (BH4) and associated neurotransmitters. BH4 is also essential for neuronal nitric oxide synthase (nNOS), so rabies is predicted to cause constriction of cerebral arteries. We assume that rabies virus, which almost exclusively targets neurons, would disproportionately affect cerebral over systemic perfusion by disrupting nNOS and lead to generalised cerebral artery spasm. Cranial artery vasospasm, therefore, was actively sought in two rabies patients, with the intention to specifically treat with BH4 and L-arginine when necessary. Flow velocities and resistive (RI) or pulsatility indices (PI) of middle cerebral arteries (MCA) were obtained by transcranial doppler ultrasound (TCD). A survival analysis of 8 attempts at the MP is presented. Of these, two cases are reported here. The first case is one child with bat-associated rabies who developed severe bilateral MCAspasm on hospital day (HD)-10 that responded to very low dose (0.2 mcg/kg/min) nitroprusside. The second case, a child with dog-associated rabies, developed spasm of MCA on HD-6 that responded to 6 mg/kg/day BH4. A second spasm with high RI (without cerebral oedema or increased intracranial pressure) responded to 20 mg/kg/day BH4 and 0.5 g/kg/dose L-arginine. Review of the TCD of the first child showed a similar second spasm seven days after first episode. Cerebral artery vasospasm occurred in the two children with rabies, but was clinically silent by standard monitoring. Spasm responded to drugs directed at the NOS pathway. Animal models for treatment of rabies are sorely needed to evaluate therapy.

Tetrahydrobiopterin availability, nitric oxide metabolism and glutathione status in the hph-1 mouse; implications for the pathogenesis and treatment of tetrahydrobiopterin deficiency states.

Tetrahydrobiopterin (BH4) is an essential cofactor for all isoforms of nitric oxide synthase. While it is well established that BH4 deficiency states are associated with impairment of dopamine, serotonin and phenylalanine metabolism, less is known with regard to the effects of deficiency of the cofactor upon nitric oxide (NO) metabolism. In this study, we have evaluated the effects of partial BH4 deficiency upon (a) tissue availability of the antioxidant glutathione, (b) basal NO production and (c) NO generation following exposure to lipopolysaccharide (LPS), which is known to increase expression of the inducible form of nitric oxide synthase. Using the hph-1 mouse, which displays a partial BH4 deficiency owing to impaired activity of GTP cyclohydrolase, we report decreased levels of glutathione in brain and kidney and evidence for decreased basal generation of nitric oxide in the periphery (as judged by the plasma nitrate plus nitrite concentration). Following LPS administration, peripheral NO generation increases. However, the concentration of plasma nitrate plus nitrite achieved was significantly decreased in the hph-1 mouse. Furthermore, LPS administration caused loss of glutathione in both wild-type and hph-1 liver and kidney. It is concluded that cofactor replacement, sufficient to fully correct a cellular BH4 deficiency, may be of benefit to patients with inborn errors of BH4 metabolism.

Max and inhibitory c-Myc mutants induce erythroid differentiation and resistance to apoptosis in human myeloid leukemia cells.

We have used the human leukemia cell line K562 as a model to study the role of c-myc in differentiation and apoptosis. We have generated stable transfectants of K562 constitutively expressing two c-Myc inhibitory mutants: D106-143, that carries a deletion in the transactivation domain of the protein, and In373, that carries an insertion in the DNA-interacting region. We show here that In373 is able to compete with c-Myc for Max binding and to inhibit the transformation activity of c-Myc. K562 cells can differentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid differentiation markers, without any detectable effects in the myelomonocytic differentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid differentiation, thus reproducing the effects of c-myc inhibitory mutants. We also studied the role of c-myc mutants and max in apoptosis of K562 induced by okadaic acid, a protein phosphatases inhibitor. The expression of D106-143 and In373 c-myc mutants and the overexpression of max reduced the apoptosis mediated by okadaic acid. The common biochemical activity of D106-143 and In373 is to bind Max and hence to titrate out c-Myc to form non-functional Myc/Max dimers. Similarly, Max overexpression would decrease the relative levels of c-Myc/Max with respect to Max/Max. The results support a model where a threshold of functional c-Myc/Max is required to maintain K562 cells in an undifferentiated state and to undergo drug-mediated apoptosis.

Purification and characterization of glycolipid transfer protein from bovine brain.

Bovine brain contains a lipid transfer protein that is specific for neutral glycosphingolipids and gangliosides but does not stimulate phospholipid or neutral lipid intermembrane transfer (Brown, R.E., Stephenson, F.A., Markello, T., Barenholz, Y. and Thompson, T.E. (1985) Chem. Phys. Lipids 38, 79-93). This report describes a new procedure for purifying glycolipid transfer protein from bovine brain as well as a characterization of the resulting protein. Chief among the newly introduced approaches are dye-ligand and fast protein cation-exchange liquid chromatography. Other modifications include increasing the overall scale of purification, incorporating a pH precipitation step and adding different proteinase inhibitors. The resulting procedure simplifies and accelerates the purification process while yielding a homogeneous protein. The purified protein has a molecular weight near 23 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chromatofocusing reveals that glycolipid transfer protein activity co-elutes with the 23 kDa protein and has an isoelectric point near pH 9.0. A similar isoelectric point is observed using denaturing isoelectric focusing conditions. The protein's amino acid composition reveals high levels of amino acids with non-polar side chains (48%). Based on the findings reported here and on previously published data, bovine brain glycolipid transfer protein has been compared to other lipid transfer proteins as well as lysosomal sphingolipid activator proteins.

Elevation of homocysteine and excitatory amino acid neurotransmitters in the CSF of children who receive methotrexate for the treatment of cancer.

PURPOSE: Folate deficiency, either by diet or drug, increases plasma homocysteine (Hcy). Hcy damages cerebrovascular endothelium, and hyperhomocysteinemia is a risk factor for stroke. Hcy is metabolized to excitatory amino acid (EAA) neurotransmitters, such as homocysteic acid (HCA) and cysteine sulfinic acid (CSA), which may cause seizures and excitotoxic neuronal death. We postulated that excess Hcy and EAA neurotransmitters may partly mediate methotrexate (MTX)-associated neurotoxicity. PATIENTS AND METHODS: In this retrospective analysis, we used high-performance liquid chromatography (HPLC) to measure Hcy, HCA, and CSA in CSF from two groups of children: (1) a control group of patients with no MTX exposure, and (2) a treatment group of patients who had received MTX no more than 7 days before a scheduled lumbar puncture. RESULTS: The treatment group had a significantly (P = .0255) greater concentration of Hcy in CSF (0.814 micromol/L +/- 0.215 [mean +/- SEM], n = 23) than the control group (0.210 micromol/L +/- 0.028, n = 34). HCA and CSA were not detected in CSF from control patients (n = 29); however, MTX caused marked accumulation of CSF HCA (119.1 micromol/L +/- 32.0, n = 16) and CSA (28.4 micromol/L +/- 7.7, n = 16) in the treatment group. Patients with neurologic toxicity at the time of lumbar puncture had many of the highest concentrations of Hcy, HCA, and CSA. CONCLUSION: These data support our hypothesis that MTX-associated neurotoxicity may be mediated by Hcy and excitotoxic neurotransmitters.

Establishing genetic interactions by a synthetic dosage lethality phenotype.

We have devised a genetic screen, termed synthetic dosage lethality, in which a cloned "reference" gene is inducibly overexpressed in a set of mutant strains carrying potential "target" mutations. To test the specificity of the method, two reference genes, CTF13, encoding a centromere binding protein, and ORC6, encoding a subunit of the origin of replication binding complex, were overexpressed in a large collection of mutants defective in either chromosome segregation or replication. CTF13 overexpression caused synthetic dosage lethality in combination with ctf14-42 (cbf2, ndc10), ctf17-61 (chl4), ctf19-58 and ctf19-26. ORC6 overexpression caused synthetic dosage lethality in combination with cdc2-1, cdc6-1, cdc14-1, cdc16-1 and cdc46-1. These relationships reflect specific interactions, as overexpression of CTF13 caused lethality in kinetochore mutants and overexpression of ORC6 caused lethality in replication mutants. In contrast, only one case of dosage suppression was observed. We suggest that synthetic dosage lethality identifies a broad spectrum of interacting mutations and is of general utility in detecting specific genetic interactions using a cloned wild-type gene as a starting point. Furthermore, synthetic dosage lethality is easily adapted to the study of cloned genes in other organisms.

Effects of AAV-2-mediated aspartoacylase gene transfer in the tremor rat model of Canavan disease.

The tremor rat is a spontaneous epilepsy model with a seizure phenotype caused by a deletion in the aspartoacylase (ASPA) gene. The absence of ASPA expression in these animals results in undetectable levels of enzyme activity and the accumulation of the substrate N-acetyl-aspartate (NAA) in brain, leading to generalized myelin vacuolation and severe motor and cognitive impairment. In support of human gene therapy for CD, recombinant adeno-associated viral vector (AAV-2) expressing ASPA was stereotactically delivered to the tremor rat brain and effects on the mutant phenotype were measured. AAV-ASPA gene transfer resulted in elevated aspartoacylase bioactivity compared to untreated mutant animals and elicited a significant decrease in the pathologically elevated whole-brain NAA levels. Assessment of motor function via quantitative rotorod testing demonstrated that rats injected with AAV-ASPA significantly improved on tests of balance and coordinated locomotion compared to animals receiving control vectors. This study provides evidence that AAV-2-mediated aspartoacylase gene transfer to the brain improves biochemical and behavioral deficits in tremor rat mutants (tm/tm) and supports the rationale of human gene transfer for Canavan disease.

Gene discovery and expression profiling in porcine Peyer's patch.

Peyer's patches of the intestinal mucosa are essential for host defense and immune regulation in the enteric system. To better understand molecular mechanisms of Peyer's patch function, we have screened for differentially expressed genes specific to Peyer's patch. cDNA libraries were created from normal Peyer's patch, immune stimulated Peyer's patch, and pooled cDNA subtracted with fibroblast RNA. From the subtracted library, 3687 expressed sequence tags (ESTs), representing 2414 unique nucleotide sequences, were isolated, identified by BLAST searches against public databases, and spotted onto a microarray for gene expression profiling. Approximately 30% of these ESTs BLAST to genes of unknown function and 20% have no known homology in the public databases (novel genes). Of the novel genes, 70% are expressed in normal immune tissues by microarray analysis, suggesting that at least 371 of the unidentified EST sequences from the subtracted library are novel porcine genes and can now be further characterized to determine their function in the porcine Peyer's patch. We surmise that the products of these genes participate in biochemical and cellular functions related to the unique immunological and gastroenterological functions of the small intestine. The BLAST and gene ontology information for each of the subtracted library EST sequences, the normal and immune stimulated libraries, and the microarray are all valuable resources that will facilitate further examination of the biological function of porcine Peyer's patch tissue.

S-adenosylmethionine levels in psychiatric and neurological disorders: a review.

INTRODUCTION: S-adenosylmethionine (SAMe) is an important methyl donor in over 35 methylation reactions involving DNA, proteins, phospholipids and catechol- and indole- amines. MATERIAL AND METHODS: This article reviews the studies that have examined brain and blood levels of SAMe in several psychological, neurological and metabolic disorders. RESULTS: Although studies have found no consistent changes in whole blood SAMe levels in psychiatric patients, other investigators have found low cerebrospinal fluid (CSF) SAMe levels in patients with neurological disorders such as Alzheimer's dementia, subacute combined degeneration of the spinal cord (SACD), and HIV-related neuropathies, as well as in patients with metabolic disorders such as 5, 10-CH2-H4 folate reductase deficiency. CONCLUSION: Intravenous or oral administration of SAMe thus represents a possible treatment for these neurological and metabolic disorders.

Aromatic L-amino acid decarboxylase deficiency: clinical features, diagnosis, and treatment of a new inborn error of neurotransmitter amine synthesis.

We report the clinical features, biochemical details, and treatment of the first detected cases of an inborn error of aromatic L-amino acid decarboxylase. Male monozygotic twins presented with extreme hypotonia and oculogyric crises. Concentrations of biogenic amines and their metabolites were reduced considerably both centrally and peripherally. Pterin and phenylalanine metabolism were normal. Activity of aromatic L-amino acid decarboxylase was virtually absent in a liver biopsy sample and greatly reduced in plasma. Concentrations of L-dopa, 3-methoxytyrosine, and 5-hydroxytryptophan were elevated in CSF, plasma, and urine. CSF S-adenosylmethionine concentrations were reduced. Pyridoxine treatment had no clinical effect but led to a fall in CSF L-dopa and 3-methoxytyrosine and a rise in S-adenosylmethionine. Treatment with either bromocriptine or tranylcypromine stopped the abnormal eye movements; tranylcypromine treatment also improved muscle tone and led to a rise in plasma norepinephrine and whole blood serotonin. Combined treatment with pyridoxine, bromocriptine, and tranylcypromine produced sustained improvement in tone and voluntary movements. The twins' parents were asymptomatic but had reduced plasma aromatic L-amino acid decarboxylase activity, consistent with heterozygosity. We monitored a subsequent pregnancy through biochemical analyses of a fetal liver biopsy sample and of amniotic fluid. We predicted an unaffected fetus, which was confirmed clinically and biochemically after birth.

Demyelination and decreased S-adenosylmethionine in 5,10-methylenetetrahydrofolate reductase deficiency.

We previously described demyelination in the brain and subacute combined degeneration of the spinal cord in a patient with 5,10-methylenetetrahydrofolate reductase deficiency. To assess the role of methionine, S-adenosylmethionine, folate, and neurotransmitter amine metabolism in the demyelination process, we measured these metabolites in CSF from this patient; the findings are compared with those obtained from three patients in whom neurologic deterioration had been halted by the administration of betaine. Folate concentrations were low, and amine and biopterin metabolism were abnormal in all patients. Methionine and S-adenosylmethionine concentrations were undetectable in the first patient. In those receiving betaine, methionine concentrations were proportional to the dose administered and S-adenosylmethionine concentrations were near normal. The results provide the first evidence for an association between defective S-adenosylmethionine metabolism and demyelination in humans.

Short-term effects of high-dose 17beta-estradiol in postmenopausal PD patients: a crossover study.

OBJECTIVE: To examine the effect of 17beta-estradiol on the severity of the cardinal signs of PD in postmenopausal women. BACKGROUND: Although the impact of estrogens on the manifestations of PD has not been subjected to rigorous study, their use is generally thought to be associated with a detrimental antidopaminergic effect. METHODS: A double-blind, placebo-controlled, two-arm crossover study of high-dose transdermal 17beta-estradiol was conducted in eight postmenopausal women with mild to moderate PD, all but one of whom exhibited levodopa-induced dyskinesias. Patients were randomized initially to either hormonal treatment or placebo for 2 weeks, followed by a 2-week washout period, and then another 2-week crossover treatment period. Active treatment employed four skin patches each releasing 0.1 mg of estradiol daily, replaced every 2 to 3 days. RESULTS: After 10 days of treatment a significant reduction was observed in the antiparkinsonian threshold dose of IV levodopa. Mean duration and magnitude of the antiparkinsonian response to threshold or high doses of levodopa were unchanged, and dyskinesia scores were unaltered during 17beta-estradiol treatment compared with placebo. No worsening in "on" time or motor ratings with estrogen treatment was documented. CONCLUSIONS: 17beta-estradiol appears to display a slight prodopaminergic (or antiparkinsonian) effect without consistently altering dyskinesias. Standard postmenopausal replacement therapy with transdermal 17beta-estradiol is likely to be well tolerated by many female parkinsonian patients.

Clinical and therapeutic observations in aromatic L-amino acid decarboxylase deficiency.

OBJECTIVES: To elucidate the phenotype in aromatic L-amino acid decarboxylase (AADC) deficiency, a rare autosomal recessive disorder of neurotransmitter synthesis, and report preliminary treatment observations with directed therapy of the associated neurotransmitter deficiencies. BACKGROUND: AADC is a required enzyme in dopamine, norepinephrine, epinephrine, and serotonin biosynthesis. Five patients have been previously reported. Responses to treatment interventions in these patients have been mixed. METHODS: Clinical and biochemical evaluation and therapeutic trials were performed in two children over a 26-month period. RESULTS: Characteristic features included axial hypotonia, hypokinesia, and athetosis, with superimposed episodes of ocular convergence spasm, oculogyric crises, dystonia, and limb rigidity. Catecholamine deficiency was manifest by ptosis, nasal congestion, paroxysmal diaphoresis, temperature instability, and blood pressure lability. Abnormal sleep, feeding difficulties, and esophageal reflux were typical. Significant therapeutic benefit was observed in one child with a combination of pergolide, trihexyphenidyl, and tranylcypromine. Preliminary trials using serotonin receptor agonists or reuptake inhibitors resulted in adverse effects. CONCLUSIONS: The movement disorder in AADC deficiency, particularly the characteristic eye movement abnormalities, should facilitate the identification of patients with this rare but possibly underrecognized disorder. Directed therapy of the underlying dopamine and norepinephrine deficiency may be beneficial in some cases.