Down-regulation of hepatic urea synthesis by oxypurines: xanthine and uric acid inhibit N-acetylglutamate synthase

We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with (15)N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or l-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K(m) for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time- and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states.     

Fate of polyoma origin of replication after its direct introduction into mice

Recently we have developed a method for direct introduction of calcium phosphate-precipitated DNA into newborn rats. To examine whether the foreign DNA can replicate, a plasmid containing a polyoma origin of replication was injected into newborn mice. The plasmid was found intact in liver and spleen and able to transform bacteria. The foreign DNA had disappeared by the seventh day after injection. Yet, the plasmid DNA containing the polyoma origin of replication had undergone replication in both the liver and the spleen.     

Metabolism of 15NH3 in Organotypic Cerebellar Explants and Cultured Astrocytes: Studies with Gas Chromatography-Mass Spectrometry

Gas chromatography-mass spectrometry was used to study the metabolism of 15NH3 in organotypic cerebellar explants and cultured astrocyte monolayers. A steady-state level of 15NH3 was present by 1 min in both systems. Steady-state labeling in L-[amide-15N] glutamine, L-[15N]alanine, L-[15N]glutamate, and L-[15N]aspartate was attained by 1 min after 15NH3 addition in the organotypic cerebellar explants and by approximately 5 min in the cultured astrocytes. No measurable 15N labeling was noted in either glycine or serine in either system.     

Effects of Ketone Bodies on Astrocyte Amino Acid Metabolism

Abstract: The effects of acetoacetate and 3-hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with ¹⁵N as a metabolic probe and gas chromatography-mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3-hydroxybutyrate (5 m M ) to the incubation medium did not alter the initial rate of appearance of [¹⁵N]glutamate in the glia, but it did inhibit transamination of glutamate to [¹⁵N]aspartate. Addition of acetoacetate also inhibited formation of [2-¹⁵N]glutamine, but 3-hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 m M ) was also associated with diminished production of [¹⁵N]aspartate and [2-¹⁵N]glutamine with [¹⁵N]glutamate as precursor. Studies with [2-¹⁵N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [¹⁵N]GABA to [2-¹⁵N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3-hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.     

Potential of Selected Canadian Plant Species for Phytoextraction of Trace Elements From Selenium-Rich Soil Contaminated by Industrial Activity

In this preliminary screening study, we tested the phytoextraction potential of nine Canadian native/well-adapted plant species on a soil highly polluted by trace elements (TE) from a copper refinery. Plant physiological parameters and soil cover index were monitored for a 12-week period. At the end of the trial, biomass yield, bioconcentration (BFC) and translocation (TF) factors for the main TE as well as phytoextraction potential were determined. Most plants were severely injured by the high pollution levels, showing symptoms of toxicity including chlorosis, mortality and very low biomass yield. However, Indian mustard showed the highest selenium extraction potential (65 mg m^?2), even under harsh growing conditions. Based on our results, tall fescue and ryegrass, which mainly stored As, Cu, Pb and Zn within roots, could be used effectively for phytostabilization.     

Teratogen screening using transcriptome profiling of differentiating human embryonic stem cells

Teratogens are substances that may cause defects in normal embryonic development while not necessarily being toxic in adults. Identification of possible teratogenic compounds has been historically beset by the species‐specific nature of the teratogen response. To examine teratogenic effects on early human development we performed non‐biased expression profiling of differentiating human embryonic and induced pluripotent stem cells treated with several drugs – ethanol, lithium, retinoic acid (RA), caffeine and thalidomide, which is known to be highly species specific. Our results point to the potency of specific teratogens and their affected tissues and pathways. Specifically, we could show that ethanol caused dramatic increase in endodermal differentiation, RA caused misregulation of neural development and thalidomide affected both these processes. We thus propose this method as a valuable addition to currently available animal screening approaches.