Metabolism of purines in cultured normal and HPRT-deficient human fibroblasts

Very low erythrocyte hypoxanthine phosphoribosyl transferase activity was found in a 6-month-old child with microcephaly, hyperuricemia, and retardation of central nervous development, who subsequently developed self-mutilation. Fibroblast cell extracts from this patient showed less than 6% of normal activity of HPRT. Major metabolic products of adenine-8-C¹⁴ by extracts from both the patient and normal subjects were identified by high-voltage electrophoresis and paper chromatography as adenosine 5-monophosphate, adenosine, and inosine. Inosine was the principal product of the fibroblast cell extracts from the first biopsy of the patient, whereas the normal fibroblast cell extracts produced predominantly adenosine. These results suggest that interconversion of the purine nucleotides plays a significant role in metabolism of these fibroblast cell extracts.This project received support from the M.R.C. grant MA-3331 and the Canadian Arthritis and Rheumatism Society.     

Role of D-tryptophan oxidase in D-tryptophan utilization by Escherichia coli.

Mutants of Escherichia coli K-12 that require L-tryptophan (trp) are normally unable to utilize D-tryptophan to fulfill their requirement. However, secondary mutations (dadR) that confer this ability can be isolated. In such strains two distinct enzymes are found to be produced at high levels: D-amino acid oxidase (EC 1.4.3.3) and D-tryptophan oxidase. A convenient assay procedure for D-tryptophan oxidase is described. The two enzymes could be distinguished on the basis of their sensitivity to inhibition by L-phenylalanine and L-tyrosine. Strains that were trp dadR could not grow with D-tryptophan in the presence of L-phenylalanine, but further mutations, Fyo, could be isolated that allowed growth under these conditions. Some of them were characterized by further increases in the level of D-tryptophan oxidase activity and a sharp decrease in D-amino acid oxidase. These kinds of Fyo mutations lay in or near the dadR gene. The substrate specificity of the two enzymes toward a large number of compounds was examined. The transamination of aromatic keto acids was investigated. In the wild-type strain only a single enzyme, transaminase A (EC 2.6.1.5), was found, and it was irreversibly activated when subjected to elevated temperatures. The present state of our knowledge on D-amino acid utilization in E. coli is summarized.     

The spatial and temporal variability of the magnetoencephalogram alpha rhythm in the normal human subject

Using one-channel SQUID biomagnetometer alpha rhythm was recorded in five healthy men and eight healthy women. Maps of distribution of maximum values of mean MEG amplitudes were analyzed. Most frequently MEG maxima predominated in the parietal parts of the head. Less frequently they were found in the occipital parts. In single instances they could be found in the posterocentral sections and on the midline. In most cases an amplitude interhemispheric asymmetry of extrema was revealed. In men the mean amplitude of the alpha-rhythm predominated more frequently on the left side and in women--on the right one. The map of distribution of mean amplitudes was stable at least during two weeks. Narrow-band filtration allowed to reveal up to three discrete amplitude modes. At definite frequencies the MEG amplitude was continuously stable during several seconds.     

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.     

Efficiency of D-tryptophan as Niacin in rats

L-tryptophan is a very important precursor of niacin in mammals. Food preparation in which proteins are exposed to an alkali and/or high temperature for a long period generate appreciable amounts of D-amino acids from racemization. The efficiency of D-tryptophan as niacin was thus investigated by using weanling rats. The availability of D-tryptophan was almost the same as that in L-tryptophan as the precursor of niacin and was 1/6 as active as niacin.     

Reaction pathway of tryptophanase degrading D-tryptophan

Summary Tryptophanase, which has the very strict stereospecificity to L-tryptophan under ordinary condition, becomes active to D-tryptophan in highly concentrated diammoniumhydrogen phosphate solution. The reaction process of D-tryptophan degradation is studied in terms of kinetics. Diammoniumhydrogen phosphate acts on tryptophanase as activator below 3.1 M, and as noncompetitive inhibitor over it. Additionally, the pathway of the reaction is provided on the basis of kinetic parameters.Abbreviations TPase tryptophanase - L-Trp L-tryptophan - D-Trp D-tryptophan - DAP diammoniumhydrogen phosphate - PLP pyridoxal 5-phosphate     

The avoidance of D-tryptophan by the nematode Caenorhabditis elegans.

In chemotactic studies employing countercurrent separation the nematode aenorhabditis elegans was found to avoid D-tryptophan with a threshold in the range 10(-4) to 10(-3) M. There was no response to L-tryptophan up to 10(-2) M although it appeared to partially inhibit the response to D-tryptophan.     

Urinary metabolites of leukotriene B4 in the human subject

Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils and is thought to play a role in a variety of inflammatory responses in humans. The metabolism of LTB4 in vitro is complex with several competing pathways of biotransformation, but metabolism in vivo, especially for normal human subjects, is poorly understood. As part of a Phase I Clinical Trial of human tolerance to LTB4, four human subjects were injected with 150 nmol/kg LTB4 with one additional subject as placebo control. The urine of the subjects was collected in two separate pools (0-6 and 7-24 h), and aliquots from these urine collections were analyzed using high performance liquid chromatography, UV spectroscopy, and negative ion electrospray ionization tandem mass spectrometry for metabolites of LTB4. In the current investigation, 11 different metabolites of LTB4 were identified in the urine from those subjects injected with LTB4, and none were present in the urine from the placebo-injected subject. The unconjugated LTB4 metabolites found in urine were structurally characterized as 18-carboxy-LTB4, 10,11-dihydro-18-carboxy-LTB4, 20-carboxy-LTB4, and 10,11-dihydro-20-carboxy-LTB4. Several glucuronide-conjugated metabolites of LTB4 were characterized including 17-, 18-, 19-, and 20-hydroxy-LTB4, 10-hydroxy-4,6,12-octadecatrienoic acid, LTB4, and 10,11-dihydro-LTB4. The amount of LTB4 glucuronide (16.7-29.4 pmol/ml) and 20-carboxy-LTB4 (18.9-30.6 pmol/ml) present in the urine of subjects injected with LTB4 was determined using an isotope dilution mass spectrometric assay before and after treatment of the urine samples with beta-glucuronidase. The urinary metabolites of LTB4 identified in this investigation were excreted in low amounts, yet it is possible that one or more of these metabolites could be used to assess LTB4 biosynthesis following activation of the 5-lipoxygenase pathway in vivo.     

The rate of early fetal growth in the human subject

Data from 354 embryos and fetuses between 20 and 200 mm crown rump length obtained by therapeutic abortion in 3 different countries were evaluated. All Danich and Hungarian and the majority of American specimens were measured immeditely after delivery in the fresh condition. In the mathematical evaluation linear regressions were calculated by the method of least squares for arbitrarily defined ranges to 20-50 mm and 50-200 mm crown rump lenghts. The material was analyzed statistically so that confidence limits could be drawn for the estimation of gestational age from crown rump length measurements. All data in the 20-50 mm range were combined, but beyond that fetal length the statistics for the Hungarian group were calculated separatley. The equation calculated to fit the data in the 20-50 mm rage is A = 46 + 0.71 L where A is gestational age in days and L is crown rump lenght in mm. The 95% confidence limits of regression are 0.57-0.83 days/mm and the correlation between gestational age and crown rump length is 0.65. Estimates of gestational age from sitting height measurements can be made + or - 15 days with 95% confidence. The equation for the combined Danish and American data in the range 50-200 mm is A = 64 + 0.41 L. The 95% confidence limits for the regression are 0.36-0.46 days mm and the correlation between gestational age and crown rump length is 0.70. Estimates of gestational age from crown rump length can be made + or - 26 days with 95% confidence. The data from the Hungarian study in the 50-200 mm sitting height range differ from those of the combined Danish and American material. The regression of days/mm (0.22) was significantly less at p. 01 level supporting the suspected bias in the Hungarian material, but the correlation between gestational age and crown rump length, 0.62, was not significantly less than that of the combined Danish and American data. Thus, if the difference in the slope was due to a bias, the bias was relatively consistent from patient to patient. Comparison of the results with those of Streeter (1920, 1951) indicates that the considerable discrepancy at the embryonic stages diminishes gradually in the fetal period and eventually becomes quite insignificant.