William A. Bubb

Singlet Oxygen–mediated Protein Oxidation: Evidence for the Formation of Reactive Side Chain Peroxides on Tyrosine Residues¶

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.

Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation

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 13C label supplied as [3‐13C]pyruvate, [2‐13C]acetate, l‐[3‐13C]lactate, or d‐[1‐13C]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. This uptake was inhibited by MeAIB. When [1‐13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.

Proton Nuclear Magnetic Resonance—Based Metabonomics for Rapid Diagnosis of Meningitis and Ventriculitis

BACKGROUND Reduction of mortality associated with bacterial meningitis and postsurgical cerebral ventriculitis is dependent on early diagnosis and institution of appropriate therapy. Metabonomics rapidly defines metabolic profiles of biological fluids through the use of high-throughput analytical techniques combined with statistical pattern recognition tools. METHODS Proton nuclear magnetic resonance (1H NMR)-based metabonomics was applied to (1) lumbar cerebrospinal fluid samples collected prospectively from a cohort of patients with bacterial, fungal, or viral meningitis and from control subjects without neurological disease and (2) ventricular cerebrospinal fluid samples from patients with ventriculitis associated with an external ventricular drain and from control subjects. 1H NMR spectra were analyzed by the unsupervised statistical method of principal components analysis. RESULTS Metabonomic analysis clearly distinguished patients with bacterial or fungal meningitis (11 patients) from patients with viral meningitis (12) and control subjects (27) and clearly distinguished patients with postsurgical ventriculitis (5) from postsurgical control subjects (10). Metabolites of microbial and host origin that were responsible for class separation were determined. Metabonomic data also correlated with the onset and course of infection in a patient with 2 episodes of bacterial ventriculitis and with response to therapy in another patient with cryptococcal meningitis. CONCLUSIONS Metabonomic analysis is rapid, requires minimal sample processing, and is not targeted to specific microbial pathogens, making the platform potentially suitable for use in the diagnostic laboratory. This pilot study indicates that metabonomic analysis of cerebrospinal fluid is feasible and a potentially more powerful diagnostic tool than conventional rapid laboratory indicators for distinguishing bacterial from viral meningitis and for monitoring therapy. This should have important implications for early management, reduced empirical use of antibiotics, and treatment duration.

Metabolite profiling of the intraerythrocytic malaria parasite Plasmodium falciparum by (1)H NMR spectroscopy.

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 1H 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 1H 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 α‐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. Copyright

Structural characterisation of the exocellular polysaccharide produced by Streptococcus thermophilus OR 901

The exocellular polysaccharide of Streptococcus thermophilus OR 901, isolated from partially deproteinised whey, is a heteropolymer of D-galactopyranose and L-rhamnopyranose residues in the molar ratio 5:2. The structure was established by methylation analysis and 1D and 2D NMR spectroscopy of the native polysaccharide, in combination with characterisation of oligosaccharide fragments, obtained by partial acid hydrolysis, using methylation analysis and 1D 1H NMR spectroscopy. The polysaccharide has a branched heptasaccharide repeating unit with the following structure: [sequence: see text]

Magnetic susceptibility: solutions, emulsions, and cells

Differences in magnetic susceptibility between various compartments in heterogeneous samples can introduce unanticipated complications to NMR spectra. On the other hand, an understanding of these effects at the level of the underlying physical principles has led to the development of several experimental techniques that provide data on cellular function that are unique to NMR spectroscopy. To illustrate some key features of susceptibility effects we present, among a more general overview, results obtained with red blood cells and a recently described model system involving diethyl phthalate in water. This substance forms a relatively stable emulsion in water and yet it has a significant solubility of ∼5 mmol L-1 at room temperature; thus, the NMR spectrum has twice as many resonances as would be expected for a simple solution. What determines the relative intensities of the two families of peaks and can their frequencies be manipulated experimentally in a predictable way? The theory used to interpret the NMR spectra from the model system and cells was first developed in the context of electrostatics nearly a century ago, and yet some of its underlying assumptions now warrant closer scrutiny. While this insight is used in a practical way in this article, the accompanying article deals with the mathematics and physics behind this new analysis.

Brain gene expression, metabolism, and bioenergetics: interrelationships in murine models of cerebral and noncerebral malaria

Malaria infection can cause cerebral symptoms without parasite invasion of brain tissue. We examined the relationships between brain biochemis¬try, bioenergetics, and gene expression in murine mod¬els of cerebral (Plasmodium berghei ANKA) and nonce¬rebral (P. berghei K173) malaria using multinuclear NMR spectroscopy, neuropharmacological approaches, and real‐time RT‐PCR. In cerebral malaria caused by P. berghei ANKA infection, we found biochemical changes consistent with increased glutamatergic activity and decreased flux through the Krebs cycle, followed by increased production of the hypoxia markers lactate and alanine. This was accompanied by compromised brain bioenergetics. There were few significant changes in expression of mRNA for metabolic enzymes or transporters or in the rate of transport of glutamate or glucose. However, in keeping with a role for endoge¬nous cytokines in malaria cerebral pathology, there was significant up‐regulation of mRNAs for TNF‐α, inter¬feron‐γ, and lymphotoxin. These changes are consis¬tent with a state of cytopathic hypoxia. By contrast, in P. berghei K173 infection the brain showed increased metabolic rate, with no deleterious effect on bioenergetics. This was accompanied by mild up‐regulation of expression of metabolic enzymes. These changes are consistent with benign hypermetabolism whose cause remains a subject of speculation.—Rae, C., McQuillan, J. A., Parekh, S. B., Bubb, W. A., Weiser, S., Balcar, V. J., Hansen, A., Ball, H., Hunt, N. H. Brain gene expression, metabolism, and bioenergetics: interrela¬tionships in murine models of cerebral and noncerebral malaria.

Metabolites released by Cryptococcus neoformans var. neoformans and var. gattii differentially affect human neutrophil function

Differences in the ability of Cryptococcus neoformans var. neoformans (CNVN) and var. gattii (CNVG) to establish localized lesions in the lungs of healthy humans remain unexplained. In this study, CNVG infection in a rat model was characterized by early neutrophil invasion into lung tissue, but phagocytosis of cryptococci was not observed. The chemical composition of non-enzymic components secreted by one strain of each variety (heat-inactivated supernatants from CNVN and CNVG, termed vns and vgs, respectively) were compared, using magnetic resonance spectroscopy. Effects on human neutrophil viability and functions at both pH 5.5 and 7.0 were investigated, as the pH of cryptococcomas was found to be 5.4-5.6 in vivo. The supernatants were similar in composition, although metabolites in vns were generally present in higher concentrations. In addition, vgs contained two novel metabolites-acetoin and dihydroxyacetone. Polyphosphate was observed in cells from both varieties and may be a source of extracellular inorganic phosphate. Superoxide production in the presence of phorbol ester was enhanced by treatment with vns and decreased by vgs. At pH 5.5, vns caused high levels of necrosis in neutrophils, as well as increased adhesion/migration through A549 lung epithelial cell monolayers. Individual supernatant components such as polyols, acetoin, dihydroxyacetone, and gamma-aminobutyric acid exhibited both pro- and anti-inflammatory properties. Overall, we found that vgs was potentially less pro-inflammatory than vns. Inhibition of neutrophil function by products of CNVG may promote survival of extracellular organisms, and local multiplication to form cryptococcomas.

Strategies for studies of neurotoxic mechanisms involving deficient transport of L-glutamate: antisense knockout in rat brain in vivo and changes in the neurotransmitter metabolism following inhibition of glutamate transport in guinea pig brain slices

This communication briefly reviews characteristics of glutamate transport in the central nervous system and is involved in the aetiology of slow neurodegenerative diseases. Data in the literature suggest that antisense oligonucleotides targeted against glutamate transporters and administered in vivo over a period of days could be used to test the hypothesis. Data from our laboratory have indicated that single intraventricular doses of antisense oligonucleotides can also results in significant reductions in the numbers of substrate binding sites associated with glutamate transporters and may even cause subtle changes in their characteristics. In order to study metabolism in brain tissue, we have used 13C-nuclear magnetic resonance spectroscopy to analyse extracts of slices of guinea pig cerebral cortex exposed to glutamate transport inhibitor L-anti,endo-methanopyrrolidine dicarboxylate (L-a,e-MPDC). The results have shown-for the first time in an experimental model that preserves the relationship between glia and neurones within the context of brain tissue-that inhibition of L-glutamate transport can exert a significant influence on neurotransmitter-related metabolism. These findings suggest that metabolic disturbances caused by deficient glutamate transport could play a significant role in the death of neurones under pathological conditions in vivo.

Alanine metabolism, transport, and cycling in the brain.

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na+‐dependent transporters with properties similar to system B0 isoform B0AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2‐aminobicyclo[2.2.1]heptane‐2‐carboxylic acid and cycloleucine (1‐aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.