Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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The "in vivo" effects of L-phenylalanine on the gluconeogenic pathway in the liver of fasted rats with experimentally induced phenylketonuria-like characteristics have been investigated. Significant increases of the fructose 6-phosphate, glucose 6-phosphate and glucose concentrations were observed. The study of the effect of L-phenylalanine on the cytoplasmic and mitochondrial redox state and energy charge showed an increase in the mitochondrial NAD+/NADH ratio while the energy charge was virtually unchanged. The effects of phenylalanine and its metabolic derivatives (phenylacetate, phenylethylamine, phenyl-lactate, o-hydroxyphenylacetate and phenylpyruvate) on the activity of lactate dehydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in rat liver have been also investigated. Phenylpyruvate inhibited the lactate dehydrogenase activity with a Ki of 5.3 mM. Phenylpyruvate also inhibited both the mitochondrial (Ki = 4 mM) and cytoplasmic (Ki = 5 mM) malate dehydrogenase activities. Phenylpyruvate, phenylacetate and o-hydroxyphenylacetate inhibited the 3-hydroxybutyrate dehydrogenase activity with Ki values of 0.7, 6.0 and 9.5 mM respectively.
Mol Cell Biochem 1977 May 31
PMID:Experimental phenylketonuria: metabolic studies in rat liver. 19 83

1. The pathogenesis of the mental retardation in phenylketonuria remains obscure. Leucocytes have proved of value in the study of other inborn errors of metabolism. The lymphocyte is a suitable model cell for the study of mammalian metabolism, because of its ability to divide in vitro in response to various stimuli. 2. We have examined the effects of phenylalanine, phenylpyruvate, phenyl-lactate and phenylacetate on the human leucocyte and the resting and phytohaemagglutinin-stimulated rabbit lymphocyte. 3. Phenylpyruvate and phenyl-lactate reduced acetate incorporation into leucocyte lipid by 38% and 48% respectively. Only phenyl-lactate reduced acetate incorporation into the resting and stimulated lymphocyte, by 20% and 34% respectively. 4. Glucose incorporation into leucocyte lipid was unaffected by phenylalanine, phenylpyruvate and phenyl-lactate. Only phenyl-lactate inhibited (46%) the production of CO2 from glucose. 5. Phenylalanine and leucine incorporation into trichloroacetic acid-insoluble material of resting and stimulated lymphocytes was inhibited by phenyl-lactate (10-42%), phenylpyruvate (27-57%) and phenylacetate (19-39%). 6. Uridine incorporation into resting and stimulated cells was inhibited by phenyl-lactate (22-26%), phenylpyruvate (42-52%) and phenylacetate (20%). 7. Thymidine incorporation into resting lymphocytes was reduced by phenyl-lactate, phenylpyruvate, phenylacetate and phenylalanine by 12-26%. Incorporation into the stimulated cell was inhibited by phenylpyruvate and phenyl-lactate (90%) and phenylacetate (66%). 8. Phenylalanine inhibited lymphocyte pyruvate kinase and phenylpyruvate inhibited citrate synthetase. 9. These results are compared with published data relating to experimental hyperphenylalaninaemia and the effects of these metabolites on nervous tissue in vitro.
Clin Sci Mol Med 1975 Oct
PMID:Effect of phenylalanine and its metabolites on the metabolism of leucocytes and lymphocytes. 123 28

Phenylketonuria (PKU) is a metabolic disorder secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). The recent creation of a mouse strain for PAH deficiency has provided an excellent model system to explore the possibility of its phenotypic correction by hepatic gene therapy. A recombinant retrovirus containing the mouse PAH cDNA under the transcriptional control of the human CMV promoter was constructed and used to transduce hepatocytes isolated from PAH-deficient mice. Viral-transduced hepatocytes produced dramatically higher levels of mouse PAH mRNA as compared to control mock-infected hepatocytes. The PAH mRNA was translated efficiently into PAH protein that is capable of converting phenylalanine to tyrosine in vitro. These results demonstrate that the PAH-deficient mouse hepatocytes can be readily reconstituted by retroviral-mediated gene transduction, which is a crucial step towards somatic gene therapy for PKU.
Somat Cell Mol Genet 1992 Jan
PMID:Reconstitution of enzymatic activity in hepatocytes of phenylalanine hydroxylase-deficient mice. 131 61

Phenylalanine, phenylpyruvate and phenylacetate produced a considerable inhibition of chick liver mevalonate 5-pyrophosphate decarboxylase while mevalonate kinase and mevalonate 5-phosphate kinase were not significantly affected. Phenolic derivatives of phenylalanine produced a similar inhibition of decarboxylase activity than that found in the presence of phenyl metabolites. The degree of inhibition was progressive with increasing concentrations of inhibitors (1.25-5.00 mM). Simultaneous supplementation of different metabolites in conditions similar to those in experimental phenylketonuria (0.25 mM each) produced a clear inhibition of liver decarboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. To our knowledge, this is the first report on the in vitro inhibition of both liver regulatory enzymes of cholesterogenesis in phenylketonuria-like conditions. Our results show a lower inhibition of decarboxylase than that of reductase but suggest an important regulatory role of decarboxylase in cholesterol synthesis.
Mol Cell Biochem 1991 Jun 26
PMID:Effect of phenylalanine derivatives on the main regulatory enzymes of hepatic cholesterogenesis. 192 6

The application of the tools of molecular biology has led to a profound increase in our current understanding of the nature of the disease states associated with defects in the phenylalanine hydroxylase (PAH) gene. Over the past decade, the PAH cDNA has been cloned and the primary structure of the PAH protein has been determined. The PAH cDNA clone has served as an invaluable probe to define the molecular structure and chromosomal location of the PAH locus in both man and other organisms. Southern analysis using the PAH cDNA as a hybridization probe has revealed the presence of numerous restriction fragment-length polymorphisms (RFLPs) in the PAH gene, which have permitted the classification of normal and mutant PAH chromosomes. RFLP analysis has also permitted the implementation of prenatal diagnosis of phenylketonuria (PKU) and other related hyperphenylalaninemic disorders. Through the use of molecular cloning and polymerase chain reaction methodologies, many molecular lesions have now been identified in the PAH gene, and their association with different PAH haplotypes and disease phenotypes can now be addressed in a rational manner. Finally, the characterization of PAH mutations has enabled the population dynamics of phenylketonuria to be examined in several different populations.
Mol Biol Med 1991 Feb
PMID:Phenylketonuria and the phenylalanine hydroxylase gene. 194 87

A novel substitution has been characterized in the phenylalanine hydroxylase (PAH) gene that is linked exclusively to mutant haplotype 6, which is prevalent in southern Europe but rare in northern and eastern Europe. It is a G-to-A transition in intron 10, 11 bases from exon 11. This substitution creates an additional AG dinucleotide, which may serve as a cryptic splice acceptor site. Individuals who bear this substitution in the homozygous state have a severe PKU phenotype with pretreatment serum phenylalanine levels over 1200 mumol/liter. The frequency and distribution of this substitution among European populations suggests two possible founding populations, one being Middle Eastern and the other Roman. The use of this substitution as a marker to identify PKU chromosomes will be an invaluable aid to carrier screening and prenatal diagnosis in populations where mutant haplotype 6 is prevalent.
Somat Cell Mol Genet 1991 May
PMID:Molecular characterization of PKU allele prevalent in southern Europe and Ireland. 204 41

Phenylketonuria (PKU) is a genetic disorder secondary to a deficiency of hepatic phenylalanine hydroxylase (PAH). Several mutations in the PAH gene have recently been reported, and linkage disequilibrium was observed between RFLP haplotypes and specific mutations. A new molecular lesion has been identified in exon 7 of the PAH gene in a Hungarian PKU patient by direct sequencing of PCR-amplified DNA. The C-to-T transition causes the substitution of Arg243 to a termination codon, and the mutant allele is associated with haplotype 4 of the PAH gene. The mutation is present in two of nine mutant haplotype 4 alleles among Eastern Europeans and is not present among Western Europeans and Asians. The rarity of this mutant allele and its restricted geographic distribution suggest that the mutational event occurred recently on a normal haplotype 4 background in Eastern Europe.
Somat Cell Mol Genet 1990 Jan
PMID:Molecular genetics of PKU in eastern Europe: a nonsense mutation associated with haplotype 4 of the phenylalanine hydroxylase gene. 230 42

Haplotype analysis of the phenylalanine hydroxylase (PAH) gene was performed on 27 chromosomes from a sample of 14 Greek phenylketonuria (PKU) probands and their parents. The majority (94%) of the 17 mutant PAH alleles are on haplotypes 1, 2 and 4, with haplotype 1 being most common. Sixty per cent of ten control PAH alleles are on haplotypes 1, 2 and 4. Haplotype 3 was not present in either group. A new MspI restriction site was found in exon 9 of a single mutant PAH allele on haplotype 7. The mutation responsible for the restriction site alteration is a T to C transition at nucleotide 1154 of the PAH cDNA, resulting in the conversion of codon 311 from leucine to proline (L311P). The same mutation has been described on a haplotype 1 allele in a German PKU patient. A single crossover event would be required to transfer this mutation from haplotype 1 to 7. Migration of this mutation from one haplotype to another by recombination cannot be distinguished from a recurrent mutation at this site.
Mol Biol Med 1989 Jun
PMID:Phenylketonuria in the Greek population. Haplotype analysis of the phenylalanine hydroxylase gene and identification of a PKU mutation. 261 49

Cells deficient in phenylalanine hydroxylase (PAH) are tyrosine auxotrophs and will not survive in tyrosine-free media. PAH activity can be constituted in cultured cells by infection with recombinant retroviruses carrying a human PAH cDNA. Mouse hepatoma cells transformed with recombinant PAH will grow in tyrosine-free media since these cells constitutively synthesize the cofactor tetrahydrobiopterin which is essential for PAH activity. NIH3T3 cells transformed with the PAH cDNA express the PAH apoenzyme, but this enzyme is inactive in vivo since these cells do not synthesize biopterin. We describe a method of selection for PAH in the fibroblast-like NIH3T3 cells involving tyrosine-free media supplemented with biopterin, reducing agents, and antioxidants. Cells transformed with the recombinant PAH gene exhibit PAH activity in culture and will grow in the biopterin-supplemented tyrosine-free media. Metabolic selection for PAH activity provides a new selectable marker for gene transfer experiments. This method is shown to be useful in the production of high titers of recombinant retroviruses carrying PAH and provides a model for experiments in somatic gene therapy of phenylketonuria.
Somat Cell Mol Genet 1987 Mar
PMID:Selection for phenylalanine hydroxylase activity in cells transformed with recombinant retroviruses. 347 Sep 52

RNA single-strand conformation polymorphism (rSSCP) is a recently developed method for detecting genetic defects. This technique requires DNA amplification with a polymerase chain reaction making use of one T7 promoter-containing primer. Amplification products are subsequently transcribed in vitro and the labelled transcripts are analysed for single-strand conformation changes. rSSCP has been applied to mutation screening of the phenylalanine hydroxylase gene and rBAT cDNA, from PKU and cystinuric patients, respectively. Experimental evidence shows that 83% and 86% of screened PKU and cystinuric mutations, respectively, give rise to detectable rSSCP signals. Thus, results obtained show that RNA single-strand conformation polymorphism analysis is generally applicable and is a suitable technique for detecting genetic disease causing mutations, both in basic research and in clinical practice.
Mol Cell Probes 1995 Jun
PMID:Molecular screening of genetic defects with RNA-SSCP analysis: the PKU and cystinuria model. 747 14


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