Revisiting the Central Metabolism of the Bloodstream Forms of Trypanosoma brucei: Production of Acetate in the Mitochondrion Is Essential for Parasite Viability

Background

The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.

Methodology/Principal Findings

A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.

Conclusions/Significance

Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.     

The Arabidopsis oligopeptidases TOP1 and TOP2 are salicylic acid targets that modulate SA‐mediated signaling and the immune response

Salicylic acid (SA) is a small phenolic molecule with hormonal properties, and is an essential component of the immune response. SA exerts its functions by interacting with protein targets; however, the specific cellular components modulated by SA and critical for immune signal transduction are largely unknown. To uncover cellular activities targeted by SA, we probed Arabidopsis protein microarrays with a functional analog of SA. We demonstrate that thimet oligopeptidases (TOPs) constitute a class of SA‐binding enzymes. Biochemical evidence demonstrated that SA interacts with TOPs and inhibits their peptidase activities to various degrees both in vitro and in plant extracts. Functional characterization of mutants with altered TOP expression indicated that TOP1 and TOP2 mediate SA‐dependent signaling and are necessary for the immune response to avirulent pathogens. Our results support a model whereby TOP1 and TOP2 act in separate pathways to modulate SA‐mediated cellular processes.     

Why and how to objectively evaluate medical gestures?

Junior physicians mainly learn during their observation in the operative room. The senior physicians evaluate them based on the same kind of observation. Knowledge transfer evaluation is thus done without quantitative methods and it mainly lies on a subjective assessment. In this paper, we present some recent techniques used to objectively evaluate medical gestures. The classical techniques used are Hidden Markov Models (HMM) or Dynamic Time Warping (DTW). Both techniques lies on the temporal analysis of the gestures. We proposed here a technique based on the arc length parametrization in order to analyze the gestures in space which is more appropriate because it gives information about the shape of the gestures independently of the chosen coordinate system.     

Ion temperature profiles along a hydrogen diagnostic beam in a TORE SUPRA tokamak plasma

The active particle diagnostic technique is used to study the ion temperature at five spatial points along the path of a hydrogen diagnostic beam. The temperature of the main ion plasma component (deuterium ions) measured by this diagnostic technique along the beam path is compared with the temperature of carbon ions (C⁺⁵). A study is made of the following characteristic features of the behavior of the ion temperature profiles T_i in various TORE SUPRA operating modes: the formation of flat and even hollow T_i profiles in ohmic discharges with q ～3 at the plasma edge, the change in T_i profiles in ergodic divertor discharges, and the difference between the temperature of the bulk ions measured by the active particle diagnostic technique and the temperature of C⁺⁵ ions in the plasma region r/a>0.5. The features revealed are explained at a qualitative level.     

Assignment by in situ hybridization of a fibroblast growth factor receptor gene to human chromosome band 10q26

Summary A 2.3-kb cDNA probe for the human bek fibroblast growth factor receptor was used to determine the chromosomal localization of the corresponding gene by in situ hybridization. The results show that this gene, a form of which is amplified in some poorly differentiated stomach cancers, is localized on chromosome region 10q26. The two previously identified fibroblast growth factor receptor genes are thus not on the same chromosome, as the related fig (fms-like gene) fibrovblast growth factor receptor gene has previously been mapped to human chromosome region 8p12.     

Capacitative calcium entry and proliferation of human osteoblast-like MG-63 cells

Abstract. Adult bone tissue is continuously being remodelled and bone mass is maintained by a balance between osteoclastic bone resorption and osteoblastic bone formation. Alteration of osteoblastic cell proliferation may account in part for lack of balance between these two processes in bone loss of osteoporosis. There is calcium (Ca²⁺) control in numerous cellular functions; however, involvement of capacitative Ca²⁺ entry (CCE) in proliferation of bone cells is less well investigated. Objectives: The study described here was aimed to investigate roles of CCE in the proliferation of osteoblast‐like MG‐63 cells. Meterials and Methods: Pharmacological characterizations of CCE were undertaken in parallel, with evaluation of the expression of transient receptor potential canonical (TRPC) channels and of cell proliferation. Results: Intracellular Ca²⁺ store depletion by thapsigargin induced CCE in MG‐63 cells; this was characterized by a rapid transient increase of intracellular Ca²⁺ followed by significant CCE, induced by conditions that stimulated cell proliferation, namely serum and platelet‐derived growth factor. Inhibitors of store‐operated Ca²⁺ channels (2‐APB and SKF‐96365) prevented CCE, while voltage‐dependent Ca²⁺ channel blockers had no effect. Expression of various TRPC channels was shown in the cells, some having been shown to be responsible for CCE. Voltage‐dependent Ca²⁺ channel blockers had no effect on osteoblast proliferation while thapsigargin, 2‐APB and SKF‐96395, inhibited it. Cell cycle analysis showed that 2‐APB and SKF‐96395 lengthen the S and G₂/M phases, which would account for the reduction in cell proliferation. Conclusions: Our results indicate that CCE, likely attributed to the activation of TRPCs, might be the main route for Ca²⁺ influx involved in osteoblast proliferation.