The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mM) glutamine to displace 13 C label sup-13 C]glucose was investigated using NMR spectroscopy.Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. Abbreviations used: ASCT-2, system ASC amino acid transporter; ATA1 and ATA2, system A amino acid transporters; EAAT, excitatory amino acid transporter; HBSS, HEPES-buffered salt solution; MeAIB, 2-(methylamino)isobutyric acid; RPMI, Roswell Park Memorial Institute Medium 1640.
Singlet oxygen (1O2) is generated by a number of enzymes as well as by UV or visible light in the presence of a sensitizer and has been proposed as a damaging agent in a number of pathologies including cataract, sunburn, and skin cancers. Proteins, and Cys, Met, Trp, Tyr and His side chains in particular, are major targets for 1O2 as a result of their abundance and high rate constants for reaction. In this study it is shown that long‐lived peroxides are formed on free Tyr, Tyr residues in peptides and proteins, and model compounds on exposure to 1O2 generated by both photochemical and chemical methods. The yield of these species is significantly enhanced in D2O and decreased by azide. Nuclear magnetic resonance and mass spectroscopic analysis of reaction mixtures, or materials separated by high‐performance liquid chromatography, are consistent with the initial formation of an (undetected) endoperoxide that undergoes rapid ring‐opening to give a hydroperoxide situated at the C1 ring‐position (i.e. para to the phenolic group). In the presence of a free α‐amino group (e.g. with free Tyr), rapid ring‐closure occurs to give an indolic hydroperoxide that decays into the corresponding alcohol, 3a‐hydroxy‐6‐oxo‐2,3,3a,6,7,7a‐hexahydro‐1H‐indole‐2‐carboxylic acid. Hydroperoxides that lack a free α‐amino group (e.g. those formed on 3‐(4‐hydroxyphenyl)propionic acid, N‐Ac‐Tyr and Tyr‐containing peptides) are longer‐lived, with half‐lives of hours to days. These species undergo slow decay at low temperatures to give the corresponding alcohol. Their rate of decay is enhanced at 37°C, or on exposure to UV light or metal ions, and gives rise to reactive radicals, via cleavage of the peroxide bond. These radicals have been characterized by electron paramagnetic resonance spin trapping. These studies demonstrate that long‐lived Tyr‐derived peroxides are formed on proteins exposed to 1O2 and that these may promote damage to other targets via further radical generation.
NMR spectroscopy was used to identify and quantify compounds in extracts prepared from mature trophozoite-stage Plasmodium falciparum parasites isolated by saponin-permeabilisation of the host erythrocyte. One-dimensional (1)H NMR spectroscopy and four two-dimensional NMR techniques were used to identify more than 50 metabolites. The intracellular concentrations of over 40 metabolites were estimated from the (1)H NMR spectra of extracts prepared by four extraction methods: perchloric acid, methanol/water, methanol/chloroform/water, and methanol alone. The metabolites quantified included: the majority of the biological alpha-amino acids; 4-aminobutyric acid; mono-, di- and tri-carboxylic acids; nucleotides; polyamines; myo-inositol; and phosphocholine and phosphoethanolamine. The parasites also contained a significant concentration (up to 12 mM) of the exogenous buffering agent, HEPES. Although the metabolite profiles obtained with each extraction method were broadly similar, perchloric acid was found to have significant advantages over the other extraction media.
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