Michael A. Becker

Stimulation of human purine synthesis de novo by fructose infusion

In order to clarify the mechanism of hyperuricemia and hyperuricosuria resulting from rapid infusion of fructose in man, the effects of an intravenous infusion of 125-200 g of fructose given over 3-4 hr on the rate of purine synthesis de novo was measured in one individual with osteoarthritis and four patients with gout. The incorporation of 1-minus 14C glycine into urinary uric acid was measured, and the pool size and turnover of urate were assessed by renal excretion of simultaneously administered 15-N urate. Fructose caused an expansion of body urate pool in all subjects, while urate turnover was increased in four. The rate of incorporation of 14-C glycine into urinary uric acid corrected for extrarenal disposal was increased in all cases (21%-430%). In two patients, rates of incorporation of 14-C glycine into urinary creatinine were increased by 10% and 11%, while rates of incorporation into uric acid were increased 84% and 159%, respectively, as a result of fructose infusion. Specific enhancement of the rate of purine synthesis de novo was suggested by these findings. The rate of infusion appeared more important than total dose in determining the magnitude of this effect. Whether the increased rate of purine synthesis was a result of direct stimulation by a fructose metabolite or was secondary to fructose-induced purine nucleotide depletion is uncertain, since the kinetics of glycine incorporation were consistent with either mechanism. Erythrocyte PP-ribose-P concentrations, however, were diminished during infusion rather than increased as might be expected if fructose infusion stimulated purine synthesis by increasing availability of this regulatory substrate.

REM sleep deprivation impairs bar-press acquisition in rats.

Abstract Brief REM sleep deprivation immediately after training temporarily retarded bar-press acquisition, abolished observational learning of the bar-press habit, and prevented adaptation to a shift from a CRF to a DRL schedule. These results support the information-processing model of REM sleep function.

Brief posttrial REM sleep deprivation impairs discrimination learning in rats

Deprivation of REM sleep (REMD) for about 3 h immediately after each training session greatly impaired brightness discrimination learning in a Y-maze. REMD beginning 2 h after training had no effect. Immediate REMD did not impair position learning in this maze. These results suggested that one function of REM sleep is the consolidation or assimilation of information for which the animal is relatively unprepared, as described by Seligman (1970). Consolidation of learning for which the rat is prepared appears to be independent of REM sleep.

Regulation of purine nucleotide synthesis. Effects of inosine on normal and hypoxantine-guanine phosphoribosyltransferase-deficient fibroblasts.

Incubation of normal and hypoxanthine-guanine phosphoribosyltransferase-deficient (mutant) human fibroblasts with inosine results in increased intracellular concentration of 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P). The magnitude of this increase is dependent on the concentration of the nucleoside and results from donation of the ribose moiety of inosine to the ribosyl phosphate moiety of PP-ribose-P through ribose phosphate intermediates. During incubation, rates of purine nucleotide synthesis de novo, estimated by incorporation of (14C) formate into formylglycinamide ribotide, are diminished in both normal and mutant cells: 5 mM inosine inhibits purine synthesis by 60-80% in normal cells and 2-20% in hypoxanthine-guanine phosphoribosyltransferase-deficient cells. The rates of purine synthesis in both normal and mutant cells are increased, however, during incubation with methylene blue at concentrations (50-100 muM) which result in more modest increases in ribose 5-phosphate and PP-ribose-P concentrations than are observed with inosine. Saturation of the PP-ribose-P amidotransferase reaction by PP-ribose-P does not appear, therefore, to explain the failure of increased PP-ribose-P concentration to stimulate the rate of purine synthesis in either type of fibroblast during incubation with inosine. Although the dissociation between PP-ribose-P concentration and the rate of purine nucleotide synthesis in normal fibroblasts incubated with inosine may be explained at least in part by an accompanying increase in intracellular concentrations of purine nucleotide feedback inhibitors, purine nucleotide concentrations are unchanged in mutant cells during incubation with inosine; these cells, in addition, show minimal (less than 3% of normal) incorporation of labeled hypoxanthine or the hypoxanthine moiety of inosine into purine nucleotides. The effect of inosine on purine synthesis de novo in hypoxanthine-guanine phosphoribosyltransferase-deficient fibroblasts is not explained in full by consideration of the concentrations of purine nucleotides and of PP-ribose-P, the factors frequently invoked as antagonistic regulators controlling the rate of this process.

REM sleep deprivation impairs serial reversal and probability maximizing in rats

Deprivation of REM sleep (REMD) for about 3 h immediately after training greatly impaired serial spatial reversal and spatial-probability maximizing. REMD beginning 3 h after training had no effect. These results are consistent with the fact that these tasks clearly distinguish the learning ability of species with REM sleep (mammals, birds) from those without REM sleep (fish). The results suggest that the greater flexibility of mammalian behavior compared to that of lower species involves a REM-sleep information-processing mechanism.

Retroactive impairment of cooperative learning by imipramine and chlordiazepoxide in rats

Pairs of rats were placed in an apparatus where their sole food source hung over an electrified grid. The current was shut off only while one rat remained on a platform out of reach of the food, thus allowing his partner to eat. Mastery of the process of taking turns at eating required about 10 daily sessions. Injection of imipramine or chlordiazepoxide a few minutes after each feeding session prevented the development of this cooperative behavior. Drug injection 3 hrs after each session had no effect. Suppression of REM sleep during the first 3 hrs after training was considered the most likely mechanism of the drug-induced impairment.

A radioisotopic method for the assay of carbamoyl phosphate in extracts of cultured human cells

Abstract Modification of a previously described radioisotopic assay (Raijman, L. (1974) Biochem. J., 138, 225–232) has permitted measurement of carbamoyl phosphate content in perchloric acid-soluble extracts of cultured human fibroblasts and lymphoblasts. The current assay utilizes excesses of purified ornithine carbamoyltransferase and [1-14C]ornithine in the enzymatic conversion of carbamoyl phosphate to [14C]citrulline, which is separated from labeled ornithine by column chromatography on an ion-exchange resin. Carbamoyl phosphate concentrations of 15 n m can be detected by this method, by means of which carbamoyl phosphate content determined in cultured human cells ranged from 0.4 to 2.1 pmol per 106 cells. Recovery of carbamoyl phosphate during sample preparation procedure exceeded 80%.

Determinants of 5-Phosphoribosyl-1-Pyrophosphate (PRPP) Synthesis in Human Fibroblasts

The synthesis of PRPP in the intact cell depends on the in vivo activity of PRPP synthetase and on the availability of its substrates, ribose-5-phosphate (R5P) and ATP. We have studied the regulation of PRPP synthesis in cultured human fibroblasts, particularly the role of intracellular inorganic phosphate (Pi) as an activator of the enzyme and the supply of R5P for the reaction. The potential pathways of R5P generation include the oxidative and nonoxidative branches of the pentose phosphate shunt (PPS), reutilization of ribose-1-phosphate released in nucleotide catabolism by nucleoside Phosphorylase, and phosphorylation of free ribose.

Methylmercaptopurine Ribonucleoside Toxicity in Human Fibroblasts: Inhibition of Phosphoribosylpyrophosphate Synthetase as Well as Amidophosphoribosyltransferase

Abundant evidence has been presented to indicate that phospho-ribosylpyrophosphate (PRPP) is the rate-limiting substrate for purine synthesis de novo (for review, see ref.1). Cultured cells derived from patients with purine overproduction have increased intracellular PRPP concentrations resulting from either increased PRPP synthesis or diminished PRPP utilization in alternative pathways (2–5). PRPP synthesis is catalyzed by PRPP synthetase (E.C. 2.7.6.1). Several distinct superactive forms of this enzyme have been described in association with increased PRPP generation and increased rates of purine synthesis de novo (3–6). Among these mutant forms of PRPP synthetase are enzymes with: increased maximal velocity of the reaction but normal sensitivity to purine nucleotide inhibition of enzyme activity (7); normal maximal velocity but altered regulatory properties (3, 6); both altered catalytic and regulatory properties (8).

Effects of inosine on purine synthesis in normal and HGPRT-deficient human fibroblasts.

1. Incubation of normal and HGPRT-deficient fibroblasts with inosine results in increased PP-ribose-P concentrations. 2. The increased PP-ribose-P concentrations are accompanied by decreased rates of purine synthesis de novo, more marked in normal cells 3. Increased purine nucleotide concentrations during incubation with inosine provide a likely explanation for the inhibition of purine synthesis in normal cells 4. The lack of accelerated purine synthesis in mutant cells under these conditions is not fully explained by consideration of PP-ribose-P and purine nucleotide concentations.