Metabolic Cooperation of Glucose and Glutamine Is Essential for the Lytic Cycle of Obligate Intracellular Parasite Toxoplasma gondii

Toxoplasma gondii is a widespread protozoan parasite infecting nearly all warm-blooded organisms. Asexual reproduction of the parasite within its host cells is achieved by consecutive lytic cycles, which necessitates biogenesis of significant energy and biomass. Here we show that glucose and glutamine are the two major physiologically important nutrients used for the synthesis of macromolecules (ATP, nucleic acid, proteins, and lipids) in T. gondii, and either of them is sufficient to ensure the parasite survival. The parasite can counteract genetic ablation of its glucose transporter by increasing the flux of glutamine-derived carbon through the tricarboxylic acid cycle and by concurrently activating gluconeogenesis, which guarantee a continued biogenesis of ATP and biomass for host-cell invasion and parasite replication, respectively. In accord, a pharmacological inhibition of glutaminolysis or oxidative phosphorylation arrests the lytic cycle of the glycolysis-deficient mutant, which is primarily a consequence of impaired invasion due to depletion of ATP. Unexpectedly, however, intracellular parasites continue to proliferate, albeit slower, notwithstanding a simultaneous deprivation of glucose and glutamine. A growth defect in the glycolysis-impaired mutant is caused by a compromised synthesis of lipids, which cannot be counterbalanced by glutamine but can be restored by acetate. Consistently, supplementation of parasite cultures with exogenous acetate can amend the lytic cycle of the glucose transport mutant. Such plasticity in the parasite''s carbon flux enables a growth-and-survival trade-off in assorted nutrient milieus, which may underlie the promiscuous survival of T. gondii tachyzoites in diverse host cells. Our results also indicate a convergence of parasite metabolism with cancer cells.     

Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis

Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD⁺/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.     

微RNA在肿瘤细胞糖代谢中的功能

大多数癌细胞产生能量是通过高速率糖酵解,然后在胞液中进行乳酸发酵。而在大多数正常细胞中,糖酵解速率相对较低,丙酮酸主要在线粒体中进行有氧氧化。即使在氧充足的条件下,快速生长的恶性肿瘤细胞进行糖酵解的速率通常要比其正常组织来源的细胞高二百多倍。微RNA（microRNA,miRNA）是一类具有转录后调控功能的非编码RNA。近年来,越来越多的研究表明,miRNA主要通过诱导缺氧环境、影响葡萄糖摄入、调节糖酵解过程中的关键酶以及乳酸去路等诸多方面参与糖代谢过程,从而在肿瘤细胞糖代谢中发挥重要作用。     

The Mitochondrion: An Integration Point of Cellular Metabolism and Signalling

In addition to efficient synthesis of ATP by oxidative phosphorylation, acquisition of the mitochondrial endosymbiont brought a whole range of new metabolic capabilities to the ancestral eukaryotic cell lineage such that the mitochondrion retains an important role in numerous anabolic and catabolic processes. While respiration dominates metabolism of the mitochondrion, this organelle is also important in the catabolism of amino acids and the provision of carbon skeletons for biosynthesis of a wide range of compounds including amino acids, vitamins, lipids, and tetrapyrroles. However, mitochondrial metabolism is best understood in the context of cellular metabolism as a whole; this is particularly true in auxotrophic organisms such as plants. For this reason understanding of the integration of mitochondrial metabolism with associated metabolic pathways in distinct cellular locations is of great importance. The examples of photorespiration, proline, cysteine, branched chain amino acid, ascorbate and folate metabolism all indicate that mitochondrial steps in these pathways are critical to their function and regulation. Moreover, the central metabolic position of the mitochondrion and its key roles in bioenergetics and redox regulation, additionally mean that it is ideally placed to act as a sensor of the biochemical status of the cell. When taken together these observations suggest that the myriad nonrespiratory functions of the mitochondria are of vast importance in the coordination of plant cellular metabolism and function.     

Rapamycin inhibits aldolase A expression during human lymphocyte activation

Rapamycin (RAPA) strongly inhibits lymphocyte activation and proliferation, but does not affect most of the activation-related gene expression at the mRNA level. In order to understand the mechanism of action of RAPA and to gain further insights in lymphocyte signalling which is impaired by RAPA, we screened for RAPA-sensitive genes using differential hybridization. The expression of human aldolase A gene was found to be inducible during T and B cell activation, and the induction was repressed by RAPA at both the mRNA and enzymatic levels. The other two important immunosuppressants, cyclosporin A and FK506, also inhibited the mitogen-induced upregulation. However, none of these three drugs inhibited the constitutive expression. There was no fluctuation of aldolase A expression during the cell cycle, and RAPA failed to block the first cell cycle after synchronization in Jurkat cells. However, the second cycle was hampered by RAPA, and this was correlated with the inhibition of aldolase A expression during this later stage. Since aldolase A is a key enzyme in glycolysis and lymphocytes mainly depend on glycolysis for energy supply, the data from this study suggest that aldolase A might be one of the downstream targets of RAPA. The inhibition of the enzyme upregulation might deprive the cells of additional supply of energy, and prevent the cells from entering an optimal status for proliferation. © 1996 Wiley-Liss, Inc.     

Some Characteristics of Central Metabolism in Acinetobacter sp. Grown on Ethanol

Ethanol-grown cells of the mutant Acinetobacter sp. strain 1NG, incapable of producing exopolysaccharides, were analyzed for the activity of enzymes of the tricarboxylic acid (TCA) cycle and some biosynthetic pathways. In spite of the presence of both key enzymes (isocitrate lyase and malate synthase) of the glyoxylate cycle, these cells also contained all enzymes of the TCA cycle, which presumably serves biosynthetic functions. This was evident from the high activity of isocitrate dehydrogenase and glutamate dehydrogenase and the low activity of 2-oxoglutarate dehydrogenase. Pyruvate was formed in the reaction catalyzed by oxaloacetate decarboxylase, whereas phosphoenolpyruvate (PEP) was synthesized by the two key enzymes (PEP carboxykinase and PEP synthase) of gluconeogenesis. The ratio of these enzymes was different in the exponential and the stationary growth phases. The addition of the C₄-dicarboxylic acid fumarate to the ethanol-containing growth medium led to a 1.5- to 2-fold increase in the activity of enzymes of the glyoxylate cycle, as well as of fumarate hydratase, malate dehydrogenase, PEP synthase, and PEP carboxykinase (the activity of the latter enzyme increased by more than 7.5 times). The data obtained can be used to improve the biotechnology of production of microbial exopolysaccharide ethapolan on C₂-substrates.     

Molecular cloning,purification, and biochemical characterization of recombinant isocitrate dehydrogenase from Streptomyces coelicolor M-145

Isocitrate dehydrogenase is a key enzyme in carbon metabolism. In this study we demonstrated that SCO7000 of Streptomyces coelicolor M-145 codes for the isocitrate dehydrogenase. Recombinant enzyme expressed in Escherichia coli had a specific activity of 25.3 μmoles/mg/min using NADP⁺ and Mn²⁺ as a cofactor, 40-times higher than that obtained in cell-free extract. Pure IDH showed a single band with an apparent Mr of 84 KDa in SDS-PAGE, which was also recognized as His-tag protein in the Western blot. Unexpectedly, in ND-PAGE conditions showed a predominant band of ~168 KDa that corresponded to the dimeric form of ScIDH. Also, zymogram assay and analytical gel filtration reveal that dimer was the active form. Kinetic parameters were 1.38, 0.11, and 0.109?mM for isocitrate, NADP, and Mn²⁺, respectively. ATP, ADP, AMP, and their mixtures were the main ScIDH activity inhibitors. Zn²⁺, Mg²⁺, Ca²⁺, and Cu⁺ had inhibitory effect on enzyme activity.     

The Enzymes of Carbon Metabolism in the Thermotolerant Bacillar Strain K1

To determine enzymatic activities in the thermotolerant strain K1 (formerly Sulfobacillus thermosulfidooxidans subsp. thermotolerans), it was grown in a mineral medium with (1) thiosulfate and Fe²⁺ or pyrite (autotrophic conditions), (2) Fe²⁺, thiosulfate, and yeast extract or glucose (mixotrophic conditions), and (3) yeast extract (heterotrophic conditions). Cells grown mixo-, hetero-, and autotrophically were found to contain enzymes of the tricarboxylic acid (TCA) cycle, as well as malate synthase, an enzyme of the glyoxylate cycle. Cells grown organotrophically in a medium with yeast extract exhibited the activity of the key enzymes of the Embden–Meyerhof–Parnas and Entner–Doudoroff pathways. The increased content of carbon dioxide (up to 5 vol %) in the auto- and mixotrophic media enhanced the activity of the enzymes involved in the terminal reactions of the TCA cycle and the enzymes of the pentose phosphate pathway. Carbon dioxide is fixed in the Calvin cycle. The highest activity of ribulose bisphosphate carboxylase was detected in cells grown autotrophically at the atmospheric content of CO₂ in the air used for aeration of the growth medium. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phospho-enolpyruvate carboxytransphosphorylase decreased with increasing content of CO₂ in the medium.     

OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ～0.4 pH units that persist in cells permeabilized in ion‐free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro‐fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro‐fission Dynamin‐related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.     

NAD~+对小麦叶片线粒体甘氨酸氧化和三羧酸代谢的调节

外源NAD~+对小麦叶片线粒体内甘氨酸、苹果酸及α—酮戊二酸氧化都有促进作用。当几种呼吸底物同时存在时,其中甘氨酸的氧化抑制了其他底物的同时氧化,因为催化这两类废物氧化的酶对NAD~+的亲和力和对NADH/NAD~+比值的敏感程度有差异,催化甘氨酸氧化的甘氨酸脱羧酶对线粒体基质内可利用的NAD~+的亲和力分别比苹果酸脱氢酶和α—酮戊二酸脱氢酶的亲和力大约1或2倍。另外,甘氨酸亦可通过保持线粒体基质内高NADH/NAD~+比值来影响三羧酸环的正常代谢。     

The pattern and kinetics of substrate metabolism of Campylobacter jejuni and Campylobacter coli

AIMS: The main aim was to investigate the patterns and kinetics of substrate oxidation by Campylobacter jejuni and C. coli. METHODS AND RESULTS: Substrate oxidation profiles by 100 strains were determined using oxygen electrode system. All the isolates tested oxidized formate, l-lactate, cysteine, glutamine and serine with high oxidation rates and high affinity but varied in their ability to oxidize citric acid cycle intermediates, aspartic acid and serine. CONCLUSIONS: Based on the oxidation ability of alpha-ketoglutarate, succinate, fumarate and aspartic acid, Campylobacter strains tested were divided into three distinct metabolic categories. The first group was able to metabolize alpha-ketoglutarate, succinate, fumarate and aspartic acid; the second group was unable to oxidize alpha-ketoglutarate; and the third group was unable to oxidize, succinate, fumarate, and aspartic acid. Furthermore, serine oxidation rate enabled the differentiation of C. jejuni and C. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall, the results highlights the extensive metabolic diversity between and within Campylobacter species. In addition, the kinetic data of oxidized substrates obtained may improve the isolation procedures of the organism.     

SDHA与肿瘤细胞代谢

细胞之间的营养竞争可以影响细胞的生长、生存和功能,不同的细胞对营养摄取条件不同,能量代谢表型各有差异,因此,细胞的状态与能量代谢是密切相关的.琥珀酸脱氢酶(SDH)位于线粒体内膜,是三羧酸循环的本质.SDH基因的突变与多种肿瘤有关.线粒体琥珀酸脱氢酶复合体是由多个亚基构成,包括SDHA、SDHB、SDHC、SDHD.其中SDHA扮演着重要的角色,SDHA突变可以引起SDH肿瘤组织中的酶活性丢失.免疫组织化学和转录组分析表明,SDHA突变会引起假性缺氧,导致血管生成增加,及其他SDHx基因突变.线粒体琥珀酸脱氢酶复合体亚单位A(SDHA)同时为线粒体电子传递链提供电子.SDHA的异常表达在肿瘤发生的过程中起到关键作用.本文从SDHA影响肿瘤细胞中能量代谢出发,对SDHA进行综述.     

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.