Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Action of Bacillus subtilis alpha-amylase on native wheat starch

Native starch granules from wheat have been subjected to enzymatic depolymerization with an alpha-amylase from Bacillus subtilis. Crystallites made from short-chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x-ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by alpha-amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.     

Pigment patterns in mutants affecting the biosynthesis of pteridines and xanthommatin in Drosophila melanogaster

Eye-color mutants of Drosophila melanogaster have been analyzed for their pigment content and related metabolites. Xanthommatin and dihydroxanthommatin (pigments causing brown eye color) were measured after selective extraction in acidified butanol. Pteridines (pigments causing red eye color) were quantitated after separation of 28 spots by thin-layer chromatography, most of which are pteridines and a few of which are fluorescent metabolites from the xanthommatin pathway. Pigment patterns have been studied in 45 loci. The pteridine pathway ramifies into two double branches giving rise to isoxanthopterin, drosopterins, and biopterin as final products. The regulatory relationship among the branches and the metabolic blockage of the mutants are discussed. The Hn locus is proposed to regulate pteridine synthesis in a step between pyruvoyltetrahydropterin and dihydropterin. The results also indicate that the synthesis and accumulation of xanthommatin in the eyes might be related to the synthesis of pteridines.Support for this work was provided to J.F. in part by a grant from the Ministerio de Universidades e Investigación (Spain) and to F.J.S. by a grant from the Ministerio de Educación y Ciencia (Spain).     

Cell Surfaces in Plant-Microorganism Interactions : VI. Elicitors of Ethylene from Colletotrichum lagenarium Trigger Chitinase Activity in Melon Plants

Treatment of melon leaves or seedlings with elicitors of Colletotrichum lagenarium, a fungal pathogen of melon, increases chitinase activity. In treated leaves, chitinase is enhanced within the first 6 hours and becomes 2 to 10 times higher than in control leaves after 24 hours. Ethylene is increased simultaneously and is correlated with chitinase elicitation. In the presence of aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, both elicitor-induced ethylene and elicitor-induced chitinase are inhibited. This inhibition is overcome by added exogenous ethylene. On the other hand, 1-aminocyclopropane-1-carboxylic acid the direct precursor of ethylene, triggers chitinase activity. Chitinase elicitation is thought to be a protein synthesis dependent process, as it does not occur in the presence of cycloheximide.     

Membrane Operational Impedance Spectra in Chara corallina Estimated by Laplace Transforms Analysis

The membrane operational impedance spectrum of Chara corallina Klein ex Willd. (R. Brown) cells is investigated using Laplace transform analysis. The spectrum changes with both amplitude and sign of the electrical stimulation when time- and voltage-dependent K⁺ channels contribute to the membrane conductance. We compare the advantages and disadvantage of this technique for studying membrane impedance with those of the alternating current method and the white noise method.     

Yeast species utilizing uric acid,adenine,n-alkylamines or diamines as sole source of carbon and energy

Yeast strains utilizing uric acid, adenine, monoamines or diamines as sole source of carbon and energy were isolated from several soil samples by the enrichment culture method. The most common species wasTrichosporon cutaneum. Strains ofCandida catenulata, C. famata, C. parapsilosis, C. rugosa, Cryptococcus laurentii, Stephanoascus ciferrii andTr. adeninovorans were also isolated. All strains utilizing uric acid as sole carbon source utilized some primaryn-alkyl-l-amines hydroxyamines or diamines as well. The ascomycetous yeast strains showing these characteristics all belonged to species known to assimilate hydrocarbons. Type strains of hydrocarbon-positive yeast species which were not found in the enrichment cultures generally assimilated putrescine, some type strains also butylamine or pentylamine, but none assimilated uric acid. Methanol-positive species were not isolated. Type strains of methanol-positive and of hydrocarbon-negative species did not assimilate uric acid, butylamine or putrescine. Assimilation of putrescine as sole source of carbon and energy may be a valuable diagnostic criterion in yeast taxonomy.     

Inhibition of cytochrome P-450 activity in rat liver microsomes by the naturally occurring flavonoid, quercetin

The kinetic characteristics and mechanism of flavonoid inhibition of cytochrome P-450-mediated reactions were examined in rat liver microsomes, using the naturally occurring flavonoid, quercetin (3,3',4',5,7-pentahydroxyflavone). Quercetin inhibited the O-deethylation of ethoxyresorufin in beta-naphthoflavone-induced microsomes by 15-80% at concentrations of 10-250 nM. The pattern of inhibition was dependent on quercetin concentration. Quercetin also inhibited p-nitroanisole demethylation and benzo(a)-pyrene hydroxylation, but did not change the proportions of the individual benzo(a)pyrene metabolites in comparison to controls. Specific steps in the P-450 reaction pathway were tested for sensitivity to quercetin inhibition. The Km values of the P-450 substrates tested were increased in the presence of quercetin; competition for and/or alteration of the substrate binding site contributes to the mechanism of inhibition. In experiments under anaerobic, carbon monoxide-saturated conditions, quercetin did not inhibit cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase. The cumene hydroperoxide-supported O-deethylation of ethoxyresorufin was inhibited by quercetin (15-60% inhibition at concentrations of 50-300 nM), suggesting that quercetin may interfere with the formation or breakdown of the oxygenated heme complex. Stoichiometry experiments established that quercetin is a potent uncoupler of P-450 reactions, elevating the rates of H2O2 formation almost twofold. Structure/activity studies indicated that certain other naturally occurring flavonoids were at least as potent inhibitors of ethoxyresorufin deethylation as quercetin. These findings are of interest in light of the significant dietary exposure of the human population to the flavonoids.     

Dietary factors affecting the urinary mutagenicity assay system. I. Detection of mutagenic activity in human urine following a fried beef meal

Studies have been conducted to determine whether the mutagens in fried beef ingested by human subjects are excreted in the urine. Urine samples were collected from individuals on liquid or regular diets before and after a fried beef meal. The mutagenic activity of the samples was tested in the Ames Salmonella/microsome assay system. The results showed that in individuals on liquid diets, most of the urinary mutagenic activity is recovered within 2-6 h after consuming a fried beef meal. In one individual tested, mutagenic activity was found in urine samples obtained 6-15 h after the fried beef meal. No mutagenic activity was detected in any of the urine samples obtained 15-24 h following the meal. In individuals on a regular diet, however, mutagenic activity was frequently observed in urine samples obtained 16-24 h following the fried beef meal, although the mutagenic activity was not as great as that in the preceding 16 h. It appears that the mutagenic agents generated by the frying of beef are ingested, absorbed, and excreted by the human body in biologically detectable quantities. These results suggest that subjects should abstain from fried beef at least one day prior to and during urine mutagenicity screening.     

Dietary factors affecting the urinary mutagenicity assay system. II. The absence of mutagenic activity in human urine following consumption of red wine or grape juice

The mutagenic activity of urine samples from nonsmoking individuals before and after the consumption of either red wine or grape juice was determined. Urine samples collected from individuals on liquid or regular diets were concentrated using XAD-2 resin. No mutagenic activity of urine concentrates was detected with Salmonella tester strains TA98 or TA100 with or without microsomal activation. The addition of 1000 units of beta-glucuronidase into the agar overlay did not show any mutagenic activity. The mutagens in red wine and grape juice, however, were extracted using the XAD-2 column. Concentrates of urine samples spiked with either of the two extracts exhibited mutagenic activity.