Heterozygosity for a point mutation in an invariant splice donor site of dihydropyrimidine dehydrogenase and severe 5-fluorouracil related toxicity.

Dihydropyrimidine dehydrogenase (DPD) is responsible for the breakdown of the widely used antineoplastic agent 5-fluorouracil (5-FU), thereby limiting the efficacy of the therapy. It has been suggested that patients suffering from 5-FU toxicities due to a low activity of DPD are genotypically heterozygous for a mutant allele of the gene encoding DPD. In this study we investigated the cDNA and a genomic region of the DPD gene of a cancer patient experiencing severe toxicity following 5-FU treatment for the presence of mutations.

Dihydropyrimidine dehydrogenase (DPD) deficiency: identification and expression of missense mutations C29R, R886H and R235W.

Dihydropyrimidine dehydrogenase (DPD) deficiency (McKusick 274270) is an autosomal recessive disease characterized by thymine-uraciluria in homozygous-deficient patients and associated with a variable clinical phenotype. Cancer patients with this defect should not be treated with the usual dose of 5-fluorouracil because of the expected lethal toxicity. In addition, heterozygosity for mutations in the DPD gene increases the risk of toxicity in cancer patients treated with this drug.

Subcellular localization of dihydropyrimidine dehydrogenase.

Although dihydropyrimidine dehydrogenase (DPD) has been purified and characterized from liver tissues of various mammals conflicting data exist on its subcellular localization. To determine the localization of DPD we prepared crude subcellular fractions of a rat liver homogenate by means of differential centrifugation. In the fractions obtained (heavy mitochondrial, light mitochondrial, microsomal and cytosolic) the activities of different marker enzymes were measured as well as the activity of DPD.

Dihydropyrimidinase deficiency and congenital microvillous atrophy: coincidence or genetic relation?

We describe a boy of consanguineous parents who suffered from intractable diarrhoea due to congenital microvillous atrophy, a recessively inherited autosomal disorder. He developed severe cholestatis starting at 2 weeks of age and leading to liver cirrhosis. His psychomotor development appeared only slightly delayed.

Identification of a four-base deletion (delTCAT296-299) in the dihydropyrimidine dehydrogenase gene with variable clinical expression.

Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting step in the breakdown of thymine, uracil, and the widely used antineoplastic drug, 5-fluorouracil. Sequence analysis of the dihydropyrimidine dehydrogenase cDNA in a Dutch consanguineous family identified a novel four-base deletion (delTCAT296-299) leading to premature termination of translation. The deletion is located in a TCAT tandem-repeat sequence and most likely results from unequal crossing-over or slipped mispairing.

MIBG causes oxidative stress and up-regulation of anti-oxidant enzymes in the human neuroblastoma cell line SK-N-BE(2c).

We report the effects of meta-iodobenzylguanidine (MIBG), a neuroblastoma-seeking agent, on cell proliferation and several oxidative stress-related parameters in the human neuroblastoma cell line SK-N-BE(2c). MIBG inhibited the proliferation of this cell line in micromolar concentrations. Measurements of the malondialdehyde (MDA) concentrations (a measure of the extent of lipid peroxidation) of cells treated with MIBG showed that increasing concentrations of MIBG led to an increase in MDA levels of the cells.

Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography.

A non-radiochemical assay procedure for CTP synthetase was developed in which CTP is detected at 280 nm after separation with anion-exchange HPLC. A complete separation of all nucleoside triphosphates was achieved within 11 min and the minimum amount of CTP which could be accurately determined proved to be 5 pmol. Therefore, our assay procedure is ten-fold more sensitive compared to the frequently used radiochemical assays.

Inborn errors of pyrimidine degradation: clinical, biochemical and molecular aspects.

The pyrimidines, uracil and thymine, are degraded in four steps. The first three steps of pyrimidine catabolism, controlled by enzyme shared by both pathways, result in the production of the neurotransmitter amino acid beta-alanine from uracil and the nonfunctional (R)-(-)-beta-aminoisobutyrate from thymine. The fourth step is controlled by several aminotransferases, which have different affinities for beta-alanine, beta-aminoisobutyrate and GABA.

The effect of the neuroblastoma-seeking agent meta-iodobenzylguanidine (MIBG) on NADH-driven superoxide formation and NADH-driven lipid peroxidation in beef heart submitochondrial particles.

In this paper we report the effects of the neuroblastoma-seeking agent meta-iodobenzylguanidine (MIBG) on NADH-driven superoxide formation and NADH-driven lipid peroxidation in beef heart submitochondrial particles. MIBG is a structural analogue of noradrenaline and is capable of inhibiting complex I and complex III of the respiratory chain. The results of our studies show that MIBG enhanced both NADH-driven superoxide formation and NADH-driven lipid peroxidation at concentrations that are likely to exist inside mitochondria of the target cells of neuroblastoma patients treated with [131I]MIBG.

HBO and the uptake and retention of [125I] MIBG in human platelets and two neuroendocrine cell lines.

In this paper we report the effects of Hyperbaric Oxygen (HBO) exposure on the uptake and retention of meta-Iodobenzylguanidine (MIBG) in human platelets and two neuroendocrine cell lines. The combination of [131I] MIBG and HBO is used for therapy of neuroblastoma. Exposure to HBO can cause oxidative stress, which is potentially capable of affecting uptake and storage of MIBG in both neuroendocrine cells and platelets.

Cell swelling and glycogen metabolism in hepatocytes from fasted rats.

Cell swelling is known to increase net glycogen production from glucose in hepatocytes from fasted rats by activating glycogen synthase. Since both active glycogen synthase and phosphorylase are present in hepatocytes, suppression of flux through phosphorylase may also contribute to the net increase in glycogen synthesis by cell swelling. We have developed an isotopic procedure to estimate the fluxes through glycogen synthase and phosphorylase in intact hepatocytes and we have examined the effect of cell swelling on both enzyme fluxes.

In-situ cytidine-deaminase activity and chromosome 1P deletion in human neuroblastoma cells.

Cytidine deaminase can cause the deamination of cytotoxic analogues of cytidine or rescue cells from the cytotoxicity of uracil analogues. Therefore, cytidine deaminase influences the cytotoxicity exerted by these compounds. We investigated the activity of this enzyme in situ in neuroblastoma cell-line cells.

Specification of the inhibitory action of MIBG on the respiratory chain by EPR scanning.

In this paper we report EPR studies on the effects of meta-Iodobenzylguanidine (MIBG), a structural analogue of norepinephrine capable of inhibiting complex I and III of the respiratory chain, on the reduction state of the various complexes of the respiratory chain in beef heart submitochondrial particles. The EPR spectrum of SMPs incubated with MIBG showed completely reduced prosthetic groups of complex I, but oxidised prosthetic groups of complex IV. This indicates that complex I is inhibited by MIBG at the end of the complex (i.

Hyperbaric oxygen enhances the effects of meta-iodobenzylguanidine (MIBG) on energy metabolism and lipid peroxidation in the human neuroblastoma cell line SK-N-BE(2C).

In this paper we report the effects of the combination of MIBG (a structural analogue of norepinephrine, used in its radio iodinated form for the diagnosis and therapy of neuroblastoma) and hyperbaric oxygen on the human neuroblastoma cell line SK-N-BE(2c). Exposure of the neuroblastoma cells to hyperbaric oxygen conditions enhanced the effects of MIBG on cell proliferation, lipid peroxidation and energy metabolism of the cell line. Cell proliferation and energy metabolism were further decreased and lipid peroxidation further increased.

Menadione inhibits MIBG uptake in two neuroendocrine cell lines.

In this paper we report on our studies of the effect of menadione on the uptake of MIBG in the neuroendocrine cell lines PC12 and SK-N-SH. Menadione inhibits the uptake of MIBG in both cell lines in a dose-dependent manner. Inhibition of MIBG uptake is most pronounced in the PC12 cell line.

Reduced replication of 3TC-resistant HIV-1 variants in primary cells due to a processivity defect of the reverse transcriptase enzyme.

Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC). These resistance mutations alter the methionine (Met) residue of the conserved YMDD motif, which is part of the catalytic core of the RT enzyme. Isoleucine (Ile) variants are initially observed, followed by the appearance and eventual outgrowth of viruses encoding valine (Val).

A point mutation in an invariant splice donor site leads to exon skipping in two unrelated Dutch patients with dihydropyrimidine dehydrogenase deficiency.

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria and associated with a variable clinical phenotype. In order to identify the molecular defect underlying complete DPD deficiency in a Dutch patient previously shown to have a 165 base pair deletion in the mature DPD mRNA, we cloned the genomic region encompassing the skipped exon and its flanking intron sequences. Sequence analysis revealed that the patient was homozygous for a single G-->A point mutation in the invariant GT dinucleotide splice donor site downstream of the skipped exon.