PLoS Pathog

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate

Patient management strategies and transplantation techniques in European stem cell transplantation centers offering breast cancer patients high-dose chemotherapy with peripheral blood stem cell support: a joint report from the EORTC and EBMT

Background and Objectives. It is increasingly being realized that there are very considerable variations in individual hospitals' strategies for managing a particular group of patients, even if using similar therapeutic regimens. Such variations make it impossible to generalize estimations of treatment costs from one setting to others. The objective of this study is to examine the extent of variation in the current approaches in Europe to peripheral blood stem cell transplantation (PBSCT) in breast carcinoma. Design and Methods. A questionnaire was developed and sent to the EBMT member institutions. The questionnaire comprised 85 questions covering the technical and clinical issues involved and the strategies followed for the management of the patients. This paper reports the results of the survey primarily by means of descriptive, univariate frequency distributions. The results of a more analytical approach, aiming at explaining patterns in the variations observed are also presented. Results. A completed questionnaire was returned by 162 centers; 60% university hospitals, 14% cancer centers and the rest general hospitals. Considerable variations are observed between the centers with respect to all aspects of patient management and technical procedures investigated. In many respects, general hospitals follow different routines from university hospitals and dedicated cancer centers. Interpretation and Conclusions. Variability to the extent observed indicates an important scope for optimization of the procedures and a large potential for reduction of costs and perhaps for improvement of outcomes. Economic evaluations, for instance comparing PBSCT with autologous BMT as support for high dose chemotherapy, cannot be generalized from one setting to another without careful examination of the procedures and strategies followed in each setting. European hospitals treating breast cancer patients with high dose chemotherapy supported by transplantation of peripheral blood stem cells use very different technical procedures for mobilization, harvest and re-implantation of stem cells. In addition, there are also wide variations in the way they manage the patients, e.g. with regard to the criteria for discharge from hospital after re-implantation. (C) 2000, Ferrata Storti Foundation

Overview of metabolites labelled from D-glucose in bloodstream form <i>T</i>. <i>brucei</i>.

<p>Metabolic network derived from detected metabolites and annotated enzymes in <i>T</i>. <i>brucei</i>. Red nodes indicate labelled metabolites from 50% U-¹³C-glucose, with darker red indicating higher percentage labelling. Grey nodes indicate no labelling detected. Nodes without spots indicate that the metabolite was not detected, but the presence is suggested by adjacent metabolites in the network.</p

Glucose is a carbon source for acetate, lipids and sugar nucleotides in WT BSF <i>T</i>. <i>brucei</i>.

<p>(A) Time-dependent incorporation of glucose into diverse pathways. Relative isotopologue abundances for unlabelled cells (0) and cells incubated with 50% U-¹³C-glucose in HMI11 for one hour (1) and 24 hours (24). Labelling reaches equilibrium within 1 hour for pyruvate, alanine, malate, aspartate and ribose 5-phosphate. Minimal labelling is observed in 2-hydroxyethyl-TPP after 1 hour, but complete (50%) labelling occurs within 24 hours, indicating a relatively slow flux towards acetate production by this pathway. (n = 3, mean ± SD). (B) Heatmap of relative isotopologue abundances for lipids after labelling for 24 hours with 50% U-¹³C-glucose in HMI11 indicating a combination of salvage mechanisms with de novo synthesized glycerol phosphate and limited incorporation of glucose-derived acetyl-CoA. After manual curation, no isotopologues containing more than 6 labelled carbons could be confirmed for the detected lipids. (C) Isotopologue abundances of UDP-N-acetyl glucosamine (UDP-GlcNAc) after labelling for 24 hours with 50% U-¹³C-glucose in HMI11 reveal incorporation of labelled carbon through glucosamine, acetate, ribose and uracil. Structures represent the likely isotopologue(s) for each mass with ¹³C atoms and bonds shown in red. Additional minor isotopologues have been omitted for clarity, the most abundant of which are isotopologues containing 3-labelled glucosamine 6-phosphate.</p

Production of succinate, pyruvate, alanine and acetate from glucose in PEPCK RNAi <i>T</i>. <i>brucei</i> measured by ¹H-NMR.

<p>^<i>a</i> Strain 427 bloodstream-form <i>T</i>. <i>brucei</i></p><p>^<i>b</i> Non-induced ^<i>RNAi</i>PEPCK-B3 (without added tetracycline)</p><p>^<i>c</i> Induced ^<i>RNAi</i>PEPCK-B3 (with added tetracycline)</p><p>Production of succinate, pyruvate, alanine and acetate from glucose in PEPCK RNAi <i>T</i>. <i>brucei</i> measured by ¹H-NMR.</p

Metabolic profile of ^<i>RNAi</i>PEPCK-B3 BSF <i>T</i>. <i>brucei</i> confirms depletion of dicarboxylic acids and pyrimidines.

<p>(A) Proton (¹H) NMR analysis of excreted pyruvate and succinate from glucose. Pyruvate (Pyr) and succinate (Suc) excreted by the bloodstream form 427 cell line (WT), and the ^<i>RNAi</i>PEPCK-B3 mutant non-induced (ni), tetracycline-induced 2 days (i 2 days), and tetracycline-induced 5 days (i 5 days) was determined by ¹H-NMR. The cells were incubated in PBS containing 4 mM glucose for 4 hours. Each spectrum corresponds to one representative experiment from a set of at least 3. A part of each spectrum ranging from 2.2 ppm to 2.4 ppm is shown. (B) LC-MS analysis of intracellular glycolytic intermediates. Relative isotopologue abundances following incubation in PBS containing 4 mM U-¹³C-glucose for 4 hours for metabolic intermediates from ^<i>RNAi</i>PEPCK-B3 mutant non-induced (NI) and tetracycline-induced for 2 days (I). Significant decreases in labelling were observed for PEPCK-derived metabolites including 3-carbon labelling in dicarboxylic acids and aspartate, and 2-carbon labelling in pyrimidine nucleotides (or 7-carbon labelling for ribose-containing metabolites). Labelling was not significantly decreased in glycolytic intermediates or in other glucose-derived metabolites, including alanine, acetate (shown here as acetyllysine) and ribose (shown here as 5-carbon labelling in UTP).</p

Labelling of pentose phosphate pathway and purine nucleotides from 50% U-13C-glucose in BSF <i>T</i>. <i>brucei</i>.

<p>(A) PPP in BSF <i>T</i>. <i>brucei</i>. Black font = detected on LCMS, grey font = inferred intermediates not detected in this assay. (B) Heatmap of relative isotopologue abundances for pentose phosphate pathway intermediates after labelling for 24 hours with 50% U-¹³C-glucose in HMI11. (C) Representative mass spectra for octulose 8-phosphate from <i>T</i>. <i>brucei</i> incubated with unlabelled (U-¹²C) glucose (top) and labelled (U-¹³C) glucose (bottom). The +3, +5 and +8 isotopologues are predominant in the labelled sample. (D) Chromatograms for all octulose 8-phosphate isotopologues from <i>T</i>. <i>brucei</i> incubated with unlabelled (U-¹²C) glucose, labelled (U-¹³C) glucose, unlabelled (U-¹²C) ribose and labelled (U-¹³C) ribose. (n = 3). (E) Heatmap of relative isotopologue abundances for purine nucleotides and cofactors after labelling for 24 hours with 50% U-¹³C-glucose in HMI11. Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; 6PG, 6-phosphogluconate; Ru5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; O8P, octulose 8-phosphate; N9P, nonulose 9-phosphate; GA3P, glyceraldehyde 3-phosphate. (F) The <i>T</i>. <i>brucei</i> transaldolase gene was heterologously expressed in <i>E</i>. <i>coli</i> and the protein purified (left hand panel, with the crude <i>E</i>. <i>coli</i> extract on the left and purified protein to the right). Purified transaldolase was shown to produce octulose 8-phosphate when provided with ribose 5-phosphate and fructose 6-phosphate as acceptor and donor substrates respectively (right hand panel). Enzymes: 1, Hexokinase; 2, glucose 6-phosphate isomerase; 17, glucose 6-phosphate dehydrogenase; 18, 6-phosphogluconate dehydrogenase; 19, ribose 5-phosphate isomerase; 20, ribokinase; 21, transaldolase.</p

Labelling of glycolytic intermediates from 50% U-¹³C-glucose in BSF <i>T</i>. <i>brucei</i>.

<p>(A) Schematic of the glycolytic pathway in BSF <i>T</i>. <i>brucei</i>. Black font = detected on LCMS, grey font = inferred intermediates not detected in this assay. (B) Heatmap of relative isotopologue abundances for glycolytic intermediates after labelling for 24 hours with 50% U-¹³C-glucose in HMI11. 0 = all carbons unlabelled (e.g. U-¹²C-glucose), 6 = six carbons labelled (e.g. U-¹³C-glucose). (C) Isotopologue abundances of hexose phosphates after labelling for 24 hours with 50% U-¹³C-glucose (n = 3, mean ± SD). (D) Isotopologue abundances of glucose 6-phosphate after labelling for 24 hours with 100% U-¹²C-glucose (white columns), 50% U-¹³C-glucose (hatched columns) or 100% U-¹³C-glucose (black columns) (n = 3, mean ± SD). Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GA3P, glyceraldehyde 3-phosphate; 1,3BPG, 1,3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; G3P, glycerol 3-phosphate. Enzymes: 1, hexokinase; 2, glucose-6-phosphate isomerase; 3, phosphofructokinase; 4, fructose-1,6-bisphosphatase; 5, aldolase; 6, triosephosphate isomerase; 7, glyceraldehyde 3-phosphate dehydrogenase; 8, phosphoglycerate kinase; 9, phosphoglycerate mutase; 10, enolase; 11, pyruvate kinase; 12, alanine aminotransferase; 13, glycerol-3-phosphate dehydrogenase; 14, glycerol kinase; 15, mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase; 16, methylglyoxal detoxification pathway.</p

PEPCK is essential to bloodstream form <i>T</i>. <i>brucei</i>.

<p>(A) Growth curve of two different clones showing down-regulation of <i>pepck</i> by RNAi (^<i>RNAi</i>PEPCK-D6 and ^<i>RNAi</i>PEPCK-B3) and the parental 427 BSF strain (WT) incubated in the presence (.i) or in the absence (.ni) of 10 μg/ml tetracycline. Cells were maintained in the exponential growth phase (between 10⁵ and 2x10⁶ cells/ml) and cumulative cell numbers have been normalized for dilution during cultivation. (B) Western blot analyses of the parental (WT) and mutant cell lines with the anti-PEPCK and anti-enolase sera containing antibodies as indicated in the left margin. (C) Western blot demonstrates significantly higher levels of PEPCK in procyclic form (PF) compared to bloodstream form (BF) WT <i>T</i>. <i>brucei</i>. *Analyses were performed on 5 x 10⁶ cells per sample and HSP-60 is shown as the loading control.</p