UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

Collection and determination of nucleotide metabolites in neonatal and adult saliva by high performance liquid chromatography with tandem mass spectrometry

Saliva contains a number of biochemical components which may be useful for diagnosis/monitoring of metabolic disorders, and as markers of cancer or heart disease. Saliva collection is attractive as a non-invasive sampling method for infants and elderly patients. We present a method suitable for saliva collection from neonates. We have applied this technique for the determination of salivary nucleotide metabolites. Saliva was collected from 10 healthy neonates using washed cotton swabs, and directly from 10 adults. Two methods for saliva extraction from oral swabs were evaluated. The analytes were then separated using high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). The limits of detection for 14 purine/pyrimidine metabolites were variable, ranging from 0.01 to 1.0 mu M. Recovery of hydrophobic purine/pyrimidine metabolites from cotton tips was consistently high using water/acetonitrile extraction (92.7-111%) compared with water extraction alone. The concentrations of these metabolites were significantly higher in neonatal saliva than in adults. Preliminary ranges for nucleotide metabolites in neonatal and adult saliva are reported. Hypoxanthine and xanthine were grossly raised in neonates (49.3 +/- 25.4; 30.9 +/- 19.5 mu M respectively) compared to adults (4.3 +/- 3.3; 4.6 +/- 4.5 mu M); nucleosides were also markedly raised in neonates. This study focuses on three essential details: contamination of oral swabs during manufacturing and how to overcome this; weighing swabs to accurately measure small saliva volumes; and methods for extracting saliva metabolites of interest from cotton swabs. A method is described for determining nucleotide metabolites using HPLC with photo-diode array or MS/MS. The advantages of utilising saliva are highlighted. Nucleotide metabolites were not simply in equilibrium with plasma, but may be actively secreted into saliva, and this process is more active in neonates than adults. (C) 2013 Elsevier B.V. All rights reserved.

Leishmania Metacyclogenesis Is Promoted in the Absence of Purines

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that "stressful conditions" will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.

Developmental disorder associated with increased cellular nucleotidase activity

Four unrelated patients are described with a syndrome that included developmental delay, seizures, ataxia, recurrent infections, severe language deficit, and an unusual behavioral phenotype characterized by hyperactivity, short attention span, and poor social interaction. These manifestations appeared within the first few years of life. Each patient displayed abnormalities on EEG. No unusual metabolites were found in plasma or urine, and metabolic testing was normal except for persistent hypouricosuria. Investigation of purine and pyrimidine metabolism in cultured fibroblasts derived from these patients showed normal incorporation of purine bases into nucleotides but decreased incorporation of uridine. De novo synthesis of purines and cellular phosphoribosyl pyrophosphate content also were moderately decreased. The distribution of incorporated purines and pyrimidines did not reveal a pattern suggestive of a deficient enzyme activity. Assay of individual enzymes in fibroblast lysates showed no deficiencies. However, the activity of cytosolic 5′-nucleotidase was elevated 6- to 10-fold. Based on the possibility that the observed increased catabolic activity and decreased pyrimidine salvage might be causing a deficiency of pyrimidine nucleotides, the patients were treated with oral pyrimidine nucleoside or nucleotide compounds. All patients showed remarkable improvement in speech and behavior as well as decreased seizure activity and frequency of infections. A double-blind placebo trial was undertaken to ascertain the efficacy of this supplementation regimen. Upon replacement of the supplements with placebo, all patients showed rapid regression to their pretreatment states. These observations suggest that increased nucleotide catabolism is related to the symptoms of these patients, and that the effects of this increased catabolism are reversed by administration of uridine.

Investigating metabolomic biomarkers of hypoxic ischaemic encephalopathy

Background An early objective biomarker to predict the severity of hypoxic-ischaemic encephalopathy (HIE) and identify infants suitable for intervention remains elusive. This thesis aims to progress metabolomic markers of HIE through a pipeline of biomarker discovery and validation by employing a novel untargeted mass spectrometry metabolomic method. Methodology Term infants with perinatal asphyxia were recruited, all having umbilical cord blood (UCB) drawn and biobanked within three hours of birth. HIE was defined by Sarnat score at 24hours and continuous multichannel-EEG. Infant neurodevelopment was assessed at 36-42 months using the Bayley Scales of Infant and Toddler Development Ed. III (BSID-III). Untargeted metabolomic analysis of UCB was performed using direct injection FT-ICR mass spectrometry (DI FT-ICR MS). Putative metabolite annotations and lipid classes were assigned and pathway analysis was performed. Results Untargeted metabolomic analysis: Thirty enrolled infants were diagnosed with HIE, including 17 mild, 8 moderate, and 5 severe cases. Pathway analysis revealed that ΔHIE was associated with a 50% and 75% perturbation of tryptophan and pyrimidine metabolism respectively, alongside alterations in amino acid pathways. Significant metabolite alterations were detected from six putatively identified lipid classes including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids and prenol lipids. Outcome prediction: Metabolite model scores significantly correlated with outcome R=0.429 (model A) and R=0.549 (model B) respectively. Model B demonstrates the potential to predict both severe outcome (AUROC of 0.915) and intact survival (AUROC of 0.800). The effect of haemolysis: On average 5% of polar and 1.5% of non-polar features were altered between paired haemolysed and clean samples. However unsupervised multivariate analysis concluded that the preanalytical variability introduced by haemolysis was negligible compared with the inherent biological inter-individual variability. Conclusion This research has employed untargeted metabolomics to identify potential early cord blood biomarkers of HIE and has performed the technical validation of previously proposed markers.

Evidence for incorporation of intact dietary pyrimidine (but not purine) nucleosides into hepatic RNA.

The absorption and metabolism of dietary nucleic acids have received less attention than those of other organic nutrients, largely because of methodological difficulties. We supplemented the rations of poultry and mice with the edible alga Spirulina platensis, which had been uniformly labeled with 13C by hydroponic culture in 13CO2. The rations were ingested by a hen for 4 wk and by four mice for 6 days; two mice were fed a normal diet and two were fed a nucleic acid-deficient diet. The animals were killed and nucleosides were isolated from hepatic RNA. The isotopic enrichment of all mass isotopomers of the nucleosides was analyzed by selected ion monitoring of the negative chemical ionization mass spectrum and the labeling pattern was deconvoluted by reference to the enrichment pattern of the tracer material. We found a distinct difference in the 13C enrichment pattern between pyrimidine and purine nucleosides; the isotopic enrichment of uniformly labeled [M + 9] isotopomers of pyrimidines exceeded that of purines [M + 10] by > 2 orders of magnitude in the avian nucleic acids and by 7- and 14-fold in the murine nucleic acids. The purines were more enriched in lower mass isotopomers, those less than [M + 3], than the pyrimidines. Our results suggest that large quantities of dietary pyrimidine nucleosides and almost no dietary purine nucleosides are incorporated into hepatic nucleic acids without hydrolytic removal of the ribose moiety. In addition, our results support a potential nutritional role for nucleosides and suggest that pyrimidines are conditionally essential organic nutrients.

In vitro expansion of U87-MG human glioblastoma cells under hypoxic conditions affects glucose metabolism and subsequent in vivo growth

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The relationship between substrate metabolism, exercise and appetite control : does glycogen availability influence the motivation to eat, energy intake or food choice?

The way in which metabolic fuels are utilised can alter the expression of behaviour in the interests of regulating energy balance and fuel availability. This is consistent with the notion that the regulation of appetite is a psychobiological process, in which physiological mediators act as drivers of behaviour. The glycogenostatic theory suggests that glycogen availability is central in eliciting negative feedback signals to restore energy homeostasis. Due to its limited storage capacity, carbohydrate availability is tightly regulated and its restoration is a high metabolic priority following depletion. It has been proposed that such depletion may act as a biological cue to stimulate compensatory energy intake in an effort to restore availability. Due to the increased energy demand, aerobic exercise may act as a biological cue to trigger compensatory eating as a result of perturbations to muscle and liver glycogen stores. However, studies manipulating glycogen availability over short-term periods (1-3 days) using exercise, diet or both have often produced equivocal findings. There is limited but growing evidence to suggest that carbohydrate balance is involved in the short-term regulation of food intake, with a negative carbohydrate balance having been shown to predict greater ad libitum feeding. Furthermore, a negative carbohydrate balance has been shown to be predictive of weight gain. However, further research is needed to support these findings as the current research in this area is limited. In addition, the specific neural or hormonal signal through which carbohydrate availability could regulate energy intake is at present unknown. Identification of this signal or pathway is imperative if a casual relationship is to be established. Without this, the possibility remains that the associations found between carbohydrate balance and food intake are incidental.

Modulation of depth-dependent properties in tissue-engineered cartilage with a semi-permeable membrane and perfusion : a continuum model of matrix metabolism and transport

The functional properties of cartilaginous tissues are determined predominantly by the content, distribution, and organization of proteoglycan and collagen in the extracellular matrix. Extracellular matrix accumulates in tissue-engineered cartilage constructs by metabolism and transport of matrix molecules, processes that are modulated by physical and chemical factors. Constructs incubated under free-swelling conditions with freely permeable or highly permeable membranes exhibit symmetric surface regions of soft tissue. The variation in tissue properties with depth from the surfaces suggests the hypothesis that the transport processes mediated by the boundary conditions govern the distribution of proteoglycan in such constructs. A continuum model (DiMicco and Sah in Transport Porus Med 50:57-73, 2003) was extended to test the effects of membrane permeability and perfusion on proteoglycan accumulation in tissue-engineered cartilage. The concentrations of soluble, bound, and degraded proteoglycan were analyzed as functions of time, space, and non-dimensional parameters for several experimental configurations. The results of the model suggest that the boundary condition at the membrane surface and the rate of perfusion, described by non-dimensional parameters, are important determinants of the pattern of proteoglycan accumulation. With perfusion, the proteoglycan profile is skewed, and decreases or increases in magnitude depending on the level of flow-based stimulation. Utilization of a semi-permeable membrane with or without unidirectional flow may lead to tissues with depth-increasing proteoglycan content, resembling native articular cartilage.

Comparison between electrically evoked and voluntary isometric contractions for biceps brachii muscle oxidative metabolism using near-infrared spectroscopy

This study compared voluntary (VOL) and electrically evoked isometric contractions by muscle stimulation (EMS) for changes in biceps brachii muscle oxygenation (tissue oxygenation index, ΔTOI) and total haemoglobin concentration (ΔtHb = oxygenated haemoglobin + deoxygenated haemoglobin) determined by near-infrared spectroscopy. Twelve men performed EMS with one arm followed 24 h later by VOL with the contralateral arm, consisting of 30 repeated (1-s contraction, 1-s relaxation) isometric contractions at 30% of maximal voluntary contraction (MVC) for the first 60 s, and maximal intensity contractions thereafter (MVC for VOL and maximal tolerable current at 30 Hz for EMS) until MVC decreased ∼30% of pre-exercise MVC. During the 30 contractions at 30% MVC, ΔTOI decrease was significantly (P < 0.05) greater and ∼tHb was significantly (P < 0.05) lower for EMS than VOL, suggesting that the metabolic demand for oxygen in EMS is greater than VOL at the same torque level. However, during maximal intensity contractions, although EMS torque (∼40% of VOL) was significantly (P < 0.05) lower than VOL, ΔTOI was similar and ΔtHb was significantly (P < 0.05) lower for EMS than VOL towards the end, without significant differences between the two sessions in the recovery period. It is concluded that the oxygen demand of the activated biceps brachii muscle in EMS is comparable to VOL at maximal intensity. © Springer-Verlag 2009.