STAT6 and STAT1 are essential antagonistic regulators of cell survival in classical Hodgkin lymphoma cell line.

Classical Hodgkin lymphoma (cHL) is a malignant lymphoid disorder characterized by aberrant activation of signaling pathways. Constitutive activation of several components of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been observed in Hodgkin and Reed/Sternberg cells, the tumor cells of cHL. In this study, we investigate the function of STAT6 in cHL cell lines and show that STAT6 promotes survival of these cells. Microarray expression analysis of STAT6-shRNA (short hairpin RNA)-expressing cHL cell lines was carried out to analyze the STAT6-mediated survival mechanism. Some of the identified genes with potentially important regulatory functions were also interleukin (IL)-4 dependently regulated in Ramos B cells and binding of STAT6 to the regulatory regions of several genes could be confirmed, indicating that these are direct STAT6 target genes. Importantly, STAT6 knockdown increased the expression and activation of STAT1 as well as the expression of known STAT1 target genes, indicating a cross-regulation between these signaling molecules. Forced expression of STAT1 was able to induce apoptosis in cHL cell line L1236. These findings indicate that both STAT6 and STAT1 can act as important antagonistic regulators in the pathogenesis of cHL.

Infantile Parkinsonism-dystonia and elevated dopamine metabolites in CSF.

Two girls and one boy are described, with severe infantile parkinsonism-dystonia. This syndrome is usually caused by endogenous dopamine deficiency but in these patients was associated with elevated dopamine metabolites in CSF and an unusual eye movement disorder: ocular flutter together with saccade initiation failure. Pyramidal tract signs also emerged in the course of the disease in two patients. This combination of symptoms and biochemical findings suggests a unique pathogenic mechanism.

Aromatic L-amino acid decarboxylase deficiency with hyperdopaminuria. Clinical and laboratory findings in response to different therapies.

Aromatic L-amino acid decarboxylase (AADC - E.C. 4.1.1.28) converts L-dopa to dopamine and 5-hydroxytryptophan to serotonin. Inherited deficiency of this enzyme leads to decreased brain levels of these neurotransmitters. Clinically this results in the development of a progressive neurometabolic disorder characterized by severe hypotonia, dystonic and choreoathetoid movements, oculogyric crises, and hypothermia from infancy. Here we describe the clinical, biochemical and molecular details of two affected brothers, one of whom, despite the lack of AADC, presented with hyperdopaminuria. In addition, we detail his reactions to treatment with dopaminergic agonists, monoamine oxidase inhibitors and pyridoxine.

Neurotransmitter metabolites in CSF: an external quality control scheme.

We report an international external quality control scheme on neurotransmitter metabolites in cerebrospinal fluid (CSF). The neurotransmitter metabolites homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) are analysed to diagnose inborn errors of neurotransmitter metabolism. HVA is the catabolite of dopamine; 5-HIAA is the catabolite of serotonin; and MHPG is the catabolite of noradrenaline. In the first phase, 12 laboratories from six countries participated in this special quality control scheme to define the present state of the art and achieve harmonization in analytical outcome and interpretation. In the second part, recoveries, dilutions and methods for sample preparation were compared. The results of 3 of 12 laboratories were excluded because of unacceptable intralaboratory coefficients of variations (CV) for HVA and/or 5-HIAA. The inter- and intralaboratory CVs, the linearity and the recovery were acceptable for the other laboratories for both parameters. Unacceptable differences in the reference ranges between laboratories, leading to differences in interpretation of the results, became obvious. There was a significant improvement of the interlaboratory CV for HVA after standardization with a calibrator. The reproducibility of MHPG measurement appeared to be adequately established in only two laboratories and recovery was low in all five measuring this metabolite. The quality control scheme is an invaluable tool for controlling the analytical outcome and providing support to laboratories to improve their quality.

Clinical and laboratory findings in twins with neonatal epileptic encephalopathy mimicking aromatic L-amino acid decarboxylase deficiency.

Aromatic L-amino acid decarboxylase (AADC) is a vitamin B 6 requiring enzyme involved in the biosynthesis of the neurotransmitters dopamine (DA) and serotonin. Lack of AADC leads to a combined deficiency of the catecholamines DA, norepinephrine (NE), epinephrine (E) as well as of serotonin. Here we describe premature twins who presented with severe seizures, myoclonus, rotatory eye movements and sudden clonic contractions. The patients showed an improvement of the clonic contractions under vitamin B 6 supplementation but died in the third week of life. In CSF and urine a biochemical pattern indicative of AADC deficiency was revealed. Concentrations of homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were decreased, in association with increased concentrations of 3-ortho-methyldopa (3-OMD) in CSF and significantly increased vanillactic acid in urine. The AADC enzyme substrates L-dopa and 5-hydroxytryptophan (5-HTP) were elevated in CSF. Elevated concentrations of threonine as well as of an unidentified compound in CSF rounded off the biochemical pattern. AADC activity was found to be increased in plasma and deficient in the liver. Molecular studies effectively ruled out a genetic defect in the AADC gene. The basis for the epileptic encephalopathy in the twins may be located in the metabolism of vitamin B 6 and remains to be defined.

beta-Ureidopropionase deficiency: a novel inborn error of metabolism discovered using NMR spectroscopy on urine.

In this work, NMR investigations that led to the discovery of a new inborn error of metabolism, beta-ureidopropionase (UP) deficiency, are reported. 1D (1)H-NMR experiments were performed using a patient's urine. 3-Ureidopropionic acid was observed in elevated concentrations in the urine spectrum. A 1D (1)H-(1)H total correlation spectroscopy (TOCSY) and two heteronuclear 2D NMR techniques (heteronuclear multiple bond correlation (HMBC) and heteronuclear single-quantum correlation (HSQC)) were used to identify the molecular structure of the compound that caused an unknown doublet resonance at 1.13 ppm. Combining the information from the various NMR spectra, this resonance could be assigned to 3-ureidoisobutyric acid. These observations suggested a deficiency of UP. With 1D (1)H-NMR spectroscopy, UP deficiency can be easily diagnosed. The (1)H-NMR spectrum can also be used to diagnose patients suffering from other inborn errors of metabolism in the pyrimidine degradation pathway.

Detection of beta-ureidopropionase deficiency with HPLC-electrospray tandem mass spectrometry and confirmation of the defect at the enzyme level.

The pyrimidine bases uracil and thymine are degraded via the consecutive action of three enzymes to beta-alanine and beta-aminoisobutyric acid, respectively. To date, a number of patients have been described with a deficiency of dihydropyrimidine dehydrogenase and dihydropyrimidinase, the first two enzymes of the pyrimidine degradation pathway. In this study, we demonstrate that the first patient presenting with N-carbamyl-beta-amino aciduria, due to a deficiency of beta-ureidopropionase, was easily diagnosed at the metabolite level using HPLC-tandem mass spectrometry. Urinary analysis showed strongly elevated levels of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid, with normal or moderately increased levels of the pyrimidine bases and the dihydropyrimidines, respectively. The deficiency of beta-ureidopropionase was confirmed by measuring all three enzymes of the pyrimidine degradation pathway. No activity of beta-ureidopropionase could be detected in a liver biopsy of the patient, while a normal activity of dihydropyrimidine dehydrogenase and dihydropyrimidinase was present. Thus, HPLC-tandem mass specrometry proved to be a powerful tool for the initial diagnosis of patients with deficiency of beta-ureidopropionase.

Tyrosine hydroxylase deficiency with severe clinical course: clinical and biochemical investigations and optimization of therapy.

Tyrosine hydroxylase deficiency was diagnosed after determination of cerebrospinal fluid neurotransmitters and DNA analysis in a child with severe axial hypotonia and hypokinesia associated with dystonic and ballistic movements. L-dopa therapy was unsuccessful, whereas a combination with selegiline, a selective monoamine oxidase-beta inhibitor, with low-dose L-dopa markedly improved the severe clinical picture.

Biochemical and molecular genetic characteristics of the severe form of tyrosine hydroxylase deficiency.

BACKGROUND: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Recently, mutations were identified in cases of autosomal recessive dopa-responsive dystonia and infantile parkinsonism. We describe a patient with severe symptoms and a new missense mutation in TH. METHODS: Relevant metabolites in urine and cerebrospinal fluid were measured by HPLC with fluorometric and electrochemical detection. All exons of the TH gene were amplified by PCR and subjected to single-strand conformation polymorphism analysis. Amplimers displaying aberrant migration patterns were analyzed by DNA sequence analysis. RESULTS: The patient presented with severe axial hypotonia, hypokinesia, reduced facial mimicry, ptosis, and oculogyric crises from infancy. The major metabolite of dopamine, homovanillic acid, was undetectable in the patient's cerebrospinal fluid. A low dose of L-dopa produced substantial biochemical but limited clinical improvement. DNA sequencing revealed a homozygous 1076G-->T missense mutation in exon 10 of the TH gene. The mutation was confirmed with restriction enzyme analysis. It was not present in 100 control alleles. Secondary structure prediction based on Chou-Fasman calculations showed an abnormal secondary structure of the mutant protein. CONCLUSIONS: We describe a new missense mutation (1076G-->T, C359F) in the TH gene. The transversion is present in all known splice variants of the enzyme. It produces more severe clinical and biochemical manifestations than previously described in TH-deficient cases. Our findings extend the clinical and the biochemical phenotype of genetically demonstrated TH deficiency.

A review of biochemical and molecular genetic aspects of tyrosine hydroxylase deficiency including a novel mutation (291delC).

An overview is given of the current knowledge on the human tyrosine hydroxylase gene and on the biochemical aspects of diagnosing defects in this gene. Diagnostic biochemical findings are described in four cases of genetically confirmed tyrosine hydroxylase deficiency. Decreased CSF levels of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), together with normal pterin and CSF tyrosine and 5-hydroxyindoleacetic acid (5-HIAA) concentrations are the diagnostic hallmarks of tyrosine hydroxylase deficiency. At the metabolite level the diagnosis can only be made reliably in CSF. Strict adherence to a standardized lumbar puncture protocol and adequate reference values are essential for diagnosis of this 'new' treatable neurometabolic disorder. Measurements of HVA, vanillylmandelic acid (VMA) or catecholamines in urine are not relevant for diagnosing tyrosine hydroxylase deficiency. The diagnosis should be considered in all children with unexplained hypokinesia and other extrapyramidal symptoms. Three of our patients are homozygous for a mutation in exon 6 (698G > A) of the tyrosine hydroxylase gene and one patient was compound heterozygous for the same mutation and a novel truncating mutation in exon 3 (291delC).

1H-NMR spectroscopy of body fluids: inborn errors of purine and pyrimidine metabolism.

BACKGROUND: The diagnosis of inborn errors of purine and pyrimidine metabolism is often difficult. We examined the potential of 1H-NMR as a tool in evaluation of patients with these disorders. METHODS: We performed 1H-NMR spectroscopy on 500 and 600 MHz instruments with a standardized sample volume of 500 microL. We studied body fluids from 25 patients with nine inborn errors of purine and pyrimidine metabolism. RESULTS: Characteristic abnormalities could be demonstrated in the 1H-NMR spectra of urine samples of all patients with diseases in the pyrimidine metabolism. In most urine samples from patients with defects in the purine metabolism, the 1H-NMR spectrum pointed to the specific diagnosis in a straightforward manner. The only exception was a urine from a case of adenine phosphoribosyl transferase deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, was not seen under the operating conditions used. Similarly, uric acid was not measured. We provide the 1H-NMR spectral characteristics of many intermediates in purine and pyrimidine metabolism that may be relevant for future studies in this field. CONCLUSION: The overview of metabolism that is provided by 1H-NMR spectroscopy makes the technique a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism.

Structures of normal single-stranded DNA and deoxyribo-3'-S-phosphorothiolates bound to the 3'-5' exonucleolytic active site of DNA polymerase I from Escherichia coli.

The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer. In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively. They serve as standards for comparison with other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis.

Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment.

BACKGROUND: Biochemical and biophysical experiments have shown that two catalytically essential divalent metal ions (termed 'A' and 'B') bind to the 3'-5' exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I. X-ray crystallographic studies have established the normal positions in the KF 3'-5' exonuclease (KF exo) active site of the two cations and the single-stranded DNA substrate. Lanthanide (III) luminescence studies have demonstrated, however, that only a single europium (III) ion (Eu3+) binds to the KF exo active site. Furthermore, Eu3+ does not support catalysis by KF exo or several other two-metal-ion phosphoryl-transfer enzymes. RESULTS: A crystal structure of KF complexed with both Eu3+ and substrate single-stranded oligodeoxynucleotide shows that a lone Eu3+ is bound near to metal-ion site A. Comparison of this structure to a relevant native structure reveals that the bound Eu3+ causes a number of changes to the KF exo active site. The scissile phosphate of the substrate is displaced from its normal position by about 1 A when Eu3+ is bound and the presence of Eu3+ in the active site precludes the binding of the essential metal ion B. CONCLUSIONS: The substantial, lanthanide-induced differences in metal-ion and substrate binding to KF exo account for the inhibition of this enzyme by Eu3+. These changes also explain the inability of KF exo to bind more than one cation in the presence of lanthanides. The mechanistic similarity between KF exo and other two-metal-ion phosphoryl-transfer enzymes suggests that the principles of lanthanide (III) ion binding and inhibition ascertained from this study will probably apply to most members of this class of enzymes.

Adenylosuccinase deficiency: possibly underdiagnosed encephalopathy with variable clinical features.

Adenylosuccinase deficiency is an autosomal recessive inherited defect of purine synthesis. In enzyme deficient patients, two normally undetectable compounds, succinylaminoimidazole carboxamide riboside and succinyladenosine, accumulate in urine, cerebrospinal fluid and, to a minor extent, in plasma. Analysing 150 highly selected urine specimens from patients with unidentified neurogenerative disorders we discovered the first two German cases of adenylosuccinase deficiency. The deficiency causes moderate to severe mental retardation, often accompanied by epileptic seizures and/or autistic features, and is occasionally associated with growth retardation and muscular hypotonia. Of the two German cases we present here, one patient fits into the clinical picture outlined by previous reports. The other patient, however, shows a pattern of symptoms so far undescribed: severe early infantile epileptic encephalopathy with reduced myelination. On mutation analysis this patient is the first to reveal a 39 base pair deletion in the adenylosuccinase gene in contrast to the point mutations detected in previous cases. Adenylosuccinase deficiency may be an underdiagnosed metabolic disorder with variable expression. This should be taken into consideration in patients with unclassified neurological conditions.

Biochemical hallmarks of tyrosine hydroxylase deficiency.

We report the biochemical hallmarks of tyrosine hydroxylase deficiency with emphasis on reliable diagnostic strategies of four new cases of an inborn error of tyrosine hydroxylase (TH). Three of our patients from different parts of the Netherlands were found homozygous for a mutation in exon 6 (G698A) of the TH gene, and one patient was found compound heterozygous for the same mutation and an additional mutation in exon 3. The first clinical symptoms of hypokinesia, rigidity of arms and legs and axial hypotonia, developed between 3 and 7 months of age. Cerebrospinal fluid investigations revealed a characteristic metabolite constellation in every case: low homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol concentrations in the presence of normal reference range 5-hydroxyindolacetic acid concentrations. Strict adherence to a standardized lumbar puncture protocol and adequate age-related reference values are essential for diagnosis of this "new" treatable neurometabolic disorder. Urinary measurements of HVA, vanillylmandelic acid, and catecholamines can lead to false-negative conclusions. All patients showed a remarkable clinical improvement on a low dose of L-dihydroxyphenylalanine/ (S)-2-(3,4-dihydroxybenzyl)-2-hydrazinpropionic acid. During treatment, cerebrospinal fluid HVA, and 3-methoxy-4-hydroxy-phenylethyleneglycol increased substantially.

Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates.

A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes. In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated. The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing. The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established. The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored. A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile. The complex with the Sp phosphorothioate at 2. 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom. Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations. Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate. Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes. These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments.

Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes.

New levels in the understanding of DNA replication have been achieved from recent crystal structure determinations of several DNA polymerases and their substrate complexes. The structure of an alpha family DNA polymerase from bacteriophage RB69 shows some similarities, but also considerable differences in structure and organization from the pol I family DNA polymerases. Also, the functions of three polymerase domains and their conserved residues have been clarified by studying structures of pol I family DNA polymerases complexed to their substrates. These structures also confirm that an identical two-metal ion catalytic mechanism proposed previously is used by both the nonhomologous pol I and pol beta family DNA polymerases.