Maria Staub

Association between Novelty Seeking and the -521 C/T polymorphism in the promoter region of the DRD4 gene.

Association between the human personality trait ‘Novelty Seeking’ and the polymorphism of the DRD4 gene was first reported by Ebstein1 and Benjamin2 in 1996. This was soon followed by replication studies in various ethnic groups and by studying the role of other neurotransmitter receptor and transporter genes in the genetic determination of human temperament. More recently, several polymorphic sites of the upstream regulatory region of the DRD4 gene have been described.3 Among these the −521 C/T single nucleotide polymorphism (SNP) was shown to be associated with the Novelty Seeking (NS) scores of the Temperament and Character Inventory (TCI) in a Japanese male population.4 We have investigated the −521 C/T SNP polymorphism in a Caucasian (Hungarian) population,5 and here we report a replication of the Japanese findings, in an association study involving 109 healthy Hungarian volunteers. We found a weak association between NS and CC vs CT or TT genotypes (P < 0.06). examination of this relation in male and female sex groups, however, strengthened the association for females (P < 0.01), but showed no genotypic effect for males.

Activation of deoxycytidine kinase by inhibition of DNA synthesis in human lymphocytes

Deoxycytidine kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-beta-D-arabinofuranosylcytosine, difluorodeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl-Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

Activation of deoxycytidine kinase by gamma-irradiation and inactivation by hyperosmotic shock in human lymphocytes

Deoxycytidine kinase (dCK) is a key enzyme in the intracellular metabolism of deoxynucleosides and their analogues, phosphorylating a wide range of drugs used in the chemotherapy of leukaemia and solid tumours. Previously, we found that activity of dCK can be enhanced by incubating primary cultures of lymphocytes with substrate analogues of the enzyme, as well as with various genotoxic agents. Here we present evidence that exposure of human lymphocytes to 0.5-2 Gy dosage of gamma-radiation as well as incubation of cells with calyculin A, a potent inhibitor of protein phosphatase 1 and 2A, both elevate dCK activity without changing the level of dCK protein. When cells were gamma-irradiated in the presence of calyculin A, a more pronounced activation of dCK was observed. In contrast, both basal and stimulated dCK activities were reduced by hyperosmotic treatment of the cells. DNA repair determined by the Comet assay and by thymidine incorporation was induced by irradiation. Complete repair of gamma-irradiated DNA was detected within 1 hr following the irradiation along with dCK activation, but the rate of repair was not accelerated by calyculin A. These data provide evidence for the activation of dCK upon DNA damage and repair that seems to be mediated by phosphorylation of the enzyme, suggesting the role of dCK in DNA repair processes.

Rapid and sensitive genotyping of dopamine D4 receptor tandem repeats by automated ultrathin-layer gel electrophoresis

Prior studies have revealed possible association between the presence of a seven repeat of the 48 bp variable number tandem repeat polymorphism of the human dopamine D4 receptor gene (DRD4) and some normal and pathological human traits, such as novelty seeking, hyperactivity disorders, and substance abuse. Some reports supported this finding whereas others did not. Incorrect genotyping could be one of the reasons for these controversial results, and might originate from preferential amplification of shorter polymerase chain reaction (PCR) products, resulting in the so‐called allele dropout. In this paper we optimized the conditions for simultaneous amplification of shorter and longer amplicons of the 48 bp repeat region of the DRD4 gene in order to avoid the loss of the longer allele and consequent incorrect genotyping, using very low DNA template concentrations and partial replacement of 2′‐deoxyguanosine‐5′‐triphosphate (dGTP) by 2′‐deoxyinosine‐5′‐triphosphate (dITP). The optimized PCR method in combination with high throughput automated ultrathin‐layer gel electrophoresis was suitable for rapid genotyping from less than a nanogram DNA using noninvasive sampling (buccal epithelial cells). All detected genotypes are presented, including such rear heterozygotes as the 2 × and 8 × 48 bp repeats in the same sample, showing the reliability of our novel detection method of longer alleles in the presence of shorter alleles.

Genotyping the -521C/T functional polymorphism in the promoter region of dopamine D4 receptor (DRD4) gene.

The ‐521C/T single nucleotide polymorphism (SNP) in the promoter region of the dopamine D4 receptor gene (DRD4) has recently been detected in oriental (Japanese) individuals and related to novelty seeking and schizophrenia. Here, we report the analysis of the ‐521C/T polymorphism in a Caucasian (Hungarian) population using two independent genotyping methods. The polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) procedure utilized the FspI restriction site around the ‐521 position. An additional, nonpolymorphic cleavage site was also included into the amplified region to serve as an internal standard for verifying the completion of the digestion. As another independent method, a tetraprimer system for single‐tube allele‐specific PCR (SAS‐PCR) was developed to generate ‐521C and ‐521T specific PCR products with different fragment sizes. Consequently, genotyping with SAS‐PCR is based on the gel‐electrophoretic separation of the allele‐specific double‐stranded DNA (dsDNA) fragments. 119 healthy Hungarian individuals were genotyped for ‐521C/T polymorphism of the dopamine D4 promoter region, using both methods. Similar allele frequencies were found (‐521C allele: 0.43; ‐521T allele: 0.57) as reported earlier for the Japanese population.

Mechanism of arginine biosynthesis in Chlamydomonas reinhardti I. Purification and properties of ornithine acetyltransferase

Abstract 1. 1. Ornithine acetyltransferase (proposed name, α-N- acetyl- l -ornithine: l -glutamate N-acetyltransferase) which catalyzes the first step in arginine biosynthesis, the formation of N-acetylglutamate from α-N- acetyl- l -ornithine and l-glutamate , has been isolated from the freshwater alga Chlamydomonas reinhardti. 2. 2. The enzyme has a broad pH optimum between 7.5 and 9. The Km value of the enzyme at pH 7.5 is 1.3 · 10−2 M for glutamate and 5.5 · 10 −3 M for α-N- acetyl- l -ornithine . The reaction is reversible; the equilibrium constant expressed as K = [acetylglutamate][ornithine]/[acetylornithine][glutamate] is 0.47. 3. 3. Beside the transferase activity, the enzyme has also a hydrolytic activity. The rate of the hydrolytic reaction for α-N acetylornithine is 1% of that of the acetyltransferase reaction. 4. 4. No specific cofactor has been found. The enzyme is inhibited by p-chloromercuribenzoate, but not by iodoacetate.

Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis

We report a novel, high‐throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele‐specific amplification with polymerase chain reaction (PCR) in a single‐step/single‐tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2—5% of the human population. The rationale of our method is the production of allele‐specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single‐tube systems, is that this method did not require the use of pre‐PCR labeled (fluorophore) primers or probes. Preferential production of the allele‐specific products was achieved by a hot‐start, time release PCR system. Specificity was increased by introducing a mismatch in the 3′‐antepenultimate position of the allele‐specific primers. This method made possible the large‐scale screening for the factor V Leiden mutation using single‐tube PCR followed by automated ultrathin‐layer agarose gel electrophoresis, with real‐time detection of the “in migratio” ethidium‐bromide‐labeled fragments.

Purification and properties of the 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K12: II. Inhibition of activity of the enzyme with phenylalanine and functional group-specific reagents

Abstract 1. 1. The inhibition of activity of the phenylalanine-sensitive 3-deoxy- d -arabino-heptulosonate-7-phosphate (DAHP) synthase (7-phospho-2-oxo-3-deoxy- d -arabino-heptonate d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) has been studied. The allosteric effector, l -phenylalanine, inhibits the activity of enzyme noncompetitively for both phosphoenolpyruvate (PEP) and erythrose 4-phosphate. Phenylalanine protects the enzyme against the inactivating effect of heat, and the Ki of enzyme for phenylalanine is 3.8 · 10 5 M. 2. 2. Bromopyruvate, an alkylating structural analog of PEP, irreversibly inactivates the enzyme, and PEP protects the enzyme against the inactivating effect of the alkylating agent. The initial step of the bromopyruvate enzyme interaction obeys the regular saturation kinetics, and the relation between bromopyruvate and PEP is competitive. The Ks of the enzyme for bromopyruvate is 2.0 mM, and the inactivation half-time of the enzyme is 1.5 min. 3. 3. DAHP synthase is a thiol enzyme. p-Hydroxymercuribenzoate reversibly, and dithiobis nitrobenzoic acid irreversibly, inactivates the enzyme. PEP protects the enzyme against the inactivating effect of 5,5′-dithio-bis-(nitrobenzoic acid). 4. 4. Metal-binding agents such as cyanide, EDTA, o-phenanthroline and 1-nitroso-2-naphthol inhibit the activity of the enzyme. The enzyme contains firmly bound heavy metal, probably Co2+.

Purification and properties of the 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (phenylalanine sensitive) of Escherichia coli K12: I. Purification of enzyme and some of its catalytic properties

Abstract 1. 1. The phenylalanine-sensitive allosteric first enzyme (isoenzyme 1a) of the aromatic amino acid biosynthetic pathway, 3- deoxy- d -arabino-heptulosonate-7-phosphate synthase ( 7- phospho-2-oxo-3-deoxy- d -arabino-heptonate d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) has been purified 160-fold from Escherichia coli K12. The molecular weight of the enzyme is 160 000. 2. 2. The enzyme has a broad pH optimum between pH 6 and 8. The initial velocity data obtained follow regular Michaelis-Menten kinetics without any detectable kinetic evidence for subunit interaction. On the basis of kinetic experiments the mechanism of enzyme action is ping-pong, and the first substrate is phosphoenol-pyruvate. The absolute Michaelis constant of the enzyme for both phosphoenol-pyruvate and erythrose 4-phosphate is 1.0 mM. Co2+, at a concentration of 1 mM, increases the enzyme activity about 2-fold. Among other ions tested, Mn2+ slightly activates the enzyme, while other heavy metal ions, such as Cu2+ and Zn2+, are inhibitory. 3. 3. Both elevated and low temperatures reversibly inactivate the enzyme. Phosphoenolpyruvate protects the enzyme against the inactivating effect of heat. The Ks of the enzyme for phosphoenolpyruvate depends on the temperature and decreases with decreasing temperature.

Deoxycytidine is salvaged not only into dna but also into phospholipid precursors

Deoxycytidine metabolism was investigated in light density human tonsillar lymphocytes using 5-3H-deoxycytidine as extracellular precursor. A significant portion of the deoxycytidine (more than 50% of ethanol soluble pool) was found to incorporate into dCDP-choline and dCDP-ethanolamine beside the well-known pathway i.e. incorporation into DNA in form of dCMP and dTMP. Hydroxyurea increased the labeling of the deoxyliponucleotides from 5-3H-deoxycytidine in spite of its inhibition of DNA synthesis.